Antigen-presenting cell-mimetic scaffolds and methods for making and using the same

ABSTRACT

Embodiments herein described provide antigen-presenting cell-mimetic scaffolds (APC-MS) and use of such scaffolds to manipulating T-cells. More specifically, the scaffolds are useful for promoting growth, division, differentiation, expansion, proliferation, activity, viability, exhaustion, anergy, quiescence, apoptosis, or death of T-cells in various settings, e.g., in vitro, ex vivo, or in vivo. Embodiments described herein further relate to pharmaceutical compositions, kits, and packages containing such scaffolds. Additional embodiments relate to methods for making the scaffolds, compositions, and kits/packages. Also described herein are methods for using the scaffolds, compositions, and/or kits in the diagnosis or therapy of diseases such as cancers, immunodeficiency disorders, and/or autoimmune disorders.

RELATED APPLICATIONS

This application is a divisional application of U.S. patent applicationSer. No. 16/316,778, filed on Jan. 10, 2019, which is a 35 U.S.C. § 371national stage filing of International Application No.PCT/US2017/041912, filed on Jul. 13, 2017, which claims priority to U.S.Provisional Patent Application No. 62/361,891, filed on Jul. 13, 2016.The entire contents of each of the aforementioned applications areexpressly incorporated herein by reference.

STATEMENT OF GOVERNMENT SUPPORT

This invention was made with Government support under Grant Nos.EB015498, EB014703, and DE013033, awarded by the U.S. NationalInstitutes of Health. The Government has certain rights in theinvention.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted electronically in XML format and is hereby incorporated byreference in its entirety. Said XML copy, created on May 19, 2023, isnamed 117823-13403 SL.xml and is 10,816 bytes in size.

BACKGROUND OF THE INVENTION

Immunotherapy involving the priming and expansion of T lymphocytes (Tcells) holds promise for the treatment of cancer and infectiousdiseases, particularly in humans (Melief et al., Immunol. Rev. 145:167-177 (1995); Riddell et al., Annu. Rev. Immunol. 13:545-586 (1995)).Current studies of adoptive transfer in patients with viral infectionsand/or cancer involve the infusion of T cells that have been stimulated,cloned and expanded for many weeks in vitro on autologous dendriticcells (DC), virally infected B cells, and/or allogeneic feeder cells(Riddell et al., Science 257:238-241 (1992); Yee et al., J. Exp. Med.192:1637-1644 (2000), Brodie et al., Nat. Med. 5:34-41 (1999); Riddellet al., Hum. Gene Ther. 3:319-338 (1992), Riddell et al., J. Immunol.Methods 128:189-201 (1990)). However, since adoptive T cellimmunotherapy clinical trials often require billions of cells (Riddellet al., 1995), existing in vitro T-cell expansion protocols are ofteninadequate to meet the demands of such trials.

Furthermore, optimal engraftment requires use of functional, and notsenescent, T-cells, at the time of re-infusion. For clinicalapplications, it is important to ensure that the T cells have thedesired functionality, i.e., that they proliferate, perform effectorfunctions and produce cytokines in a desirable manner (Liebowitz et al.,Current Opinion Oncology, 10, 533-541, 1998). In the natural setting, Tcell activation is initiated by the engagement of the T cellreceptor/CD3 complex (TCR/CD3) by a peptide-antigen bound to a majorhistocompatibility complex (MHC) molecule on the surface of anantigen-presenting cell (APC) (Schwartz, Science 248:1349 (1990)). Whilethis is the primary signal in T cell activation, other receptor-ligandinteractions between APCs and T cells are also required for completeactivation. For example, TCR stimulation in the absence of othermolecular interactions can induce a state of anergy, such that thesecells cannot respond to full activation signals upon re-stimulation(Schwartz, 1990; Harding, et al., Nature 356:607, 1992; Dudley et al.,Clinical Cancer Research., 16, 6122-6131, 2010; Rosenberg et al.,Clinical Cancer Research., 17, 4550-4557, 2011). In the alternative, Tcells may die by programmed cell death (apoptosis) when activated by TCRengagement alone (Webb et al., Cell 63:1249, 1990; Kawabe et al., Nature349:245, 1991; Kabelitz et al., Int. Immunol. 4:1381, 1992; Groux etal., Eur J. Immunol. 23:1623, 1993).

Accordingly, optimal functionality may be conferred via use of a secondsignaling molecule, e.g., a membrane-bound protein or a secreted productof the APC. In the context of membrane-bound proteins, such secondaryinteractions are usually adhesive in nature, reinforcing the contactbetween the two cells (Springer et al., Ann. Rev. Immunol. 5:223, 1987).Other signaling molecules, such as transduction of additional activationsignals from the APC to the T cell may also be involved (Bierer et al.,Adv. Cancer Res. 56:49, 1991)). For example, CD28 is a surface,glycoprotein present on 80% of peripheral T cells in humans and ispresent on both resting and activated T cells. CD28 binds to B7-1 (CD80)or B7-2 (CD86) and is one of the most potent of the known co-stimulatorymolecules (June et al., Immunol. Today 15:321 (1994), Linsley et al.,Ann. Rev. Immunol. 11:191 (1993)). CD28 ligation on T cells inconjunction with TCR engagement induces the production of interleukin-2(IL-2) (June et al., 1994; Jenkins et al., 1993; Schwartz, 1992).Secreted IL-2 is an important factor for ex vivo T cell expansion (Smithet al., Ann. N.Y. Acad. Sci. 332:423-432 (1979); Gillis et al., Nature268:154-156 (1977)).

Co-stimulation of T cells has been shown to affect multiple aspects of Tcell activation (June et al., 1994). It lowers the concentration ofanti-CD3 required to induce a proliferative response in culture (Gimmiet al., Proc. Natl. Acad. Sci. USA 88:6575 (1991)). CD28 co-stimulationalso markedly enhances the production of lymphokines by helper T cellsthrough transcriptional and post-transcriptional regulation of geneexpression Lindsten et al., Science 244:339 (1989); Fraser et al.,Science 251:313 (1991)), and can activate the cytolytic potential ofcytotoxic T cells. Inhibition of CD28 co-stimulation in vivo can blockxenograft rejection, and allograft rejection is significantly delayed(Lenschow et, al., Science 257:789 (1992); Turka et al., Proc. Natl.Acad. Sci. USA 89:11102 (1992)).

More importantly, the aforementioned effectors forstimulatory/co-stimulatory simulation have been widely applied in thecontext of manipulation of T-cells in vitro. In this context, acombination of anti-CD3 monoclonal antibody (first signal) and anti-CD28monoclonal antibody (second signal) is most commonly used to simulatethe APCs. The signals provided by anti-CD3 and anti-CD28 monoclonalantibodies are best-delivered to T-cells when the antibodies areimmobilized on a solid surface such as plastic plates (Baroja et al.,Cellular Immunology, vol. 120, 205-217, 1989; Damle et al., The Journalof Immunology, vol. 143, 1761-1767, 1989) or sepharose beads (Andersonet al., Cellular Immunology, vol. 115, 246-256, 1988). See also U.S.Pat. No. 6,352,694 issued to June et al.

A variety of surfaces and reagents containing anti-CD3 and anti-CD28monoclonal antibodies have been developed for obtaining and expanding Tcells for various applications. For instance, Levine et al. (The Journalof Immunology, vol. 159, No. 12: pp. 5921-5930, 1997) disclosetosyl-activated paramagnetic beads with a 4.5 micron (μM) diametercontaining anti-CD3 and anti-CD28 monoclonal antibodies, which can beutilized to stimulate and proliferate T-cells and induce them to producepro-inflammatory cytokines. It has also been shown that T-cellsactivated with these beads exhibit properties, such as cytokineproduction, that make them potentially useful for adoptive immunotherapy(Garlie et al., J Immunother 22(4): 336-45, 1999; Shibuya et al., ArchOtolaryngol Head Neck Surg, vol. 126, No. 4: 473-479, 2000). These beadsare commercially available from Thermo-Fisher Scientific, Inc. under thetrade name DYNABEADS CD3/CD28 T-cell expansion.

The use of paramagnetic beads with immobilized monoclonal antibodies forexpansion of T-cells in cell therapy requires separation and removal ofthe beads from the T-cells prior to patient infusion. This is a verylabor-intensive process and results in cell loss, cell damage, increasedrisk of contamination and increased cost of processing. Because of thetight association of the immobilized monoclonal antibodies on the beadswith the corresponding ligands on the surface of the target T-cells, theremoval of the beads from the T-cells is difficult. The bead-cellconjugates are often separated by waiting until the T-cells internalizethe target antigens and then using mechanical disruption techniques toseparate the beads from the T-cells. This technique can cause damage tothe T-cells and can also cause the ligated antigens on the T-cells to beremoved from the cell surface (Rubbi et al., Journal of ImmunologyMethods, 166, 233-241, 1993). In addition, since activated T-cells areoften most-desired for use in cell therapy protocols and the desirableproperties of the cells are lost during the 24-72 hour waiting time,paramagnetic separation has a limited use in the adoptive cell-therapysetting.

Techniques for separation and purification of cells attached toparamagnetic beads are also unusable in the clinical context. Forinstance, the process of removing the paramagnetic beads afterseparation from the T-cells requires the passing of the cell/beadsolution over a magnet. This process, while greatly reducing the numberof beads remaining with the T-cells, does not completely eliminate thebeads. Implantation of compositions containing beads into patients cancause toxic effects. The bead removal process also reduces the number ofT-cells available for therapy, as many T-cells remain associated withthe paramagnetic beads, even after mechanical disassociation. Some cellloss also occurs with respect to the T-cells that are manipulated butotherwise not bound to the beads because these cells are washed awayprior to the internalization and/or mechanical removal step(s).

There is, therefore, an unmet need for compositions and methods thatallow isolation of T-cells, which can be readily utilized for thetherapy of human diseases, such as immunodeficiency disorders,autoimmune disorders, and cancers. Embodiments of the instant invention,which are described in detail below, address these needs.

SUMMARY OF THE INVENTION

The present invention provides compositions and methods formanipulating, e.g., activating, stimulating, expanding, proliferating,or energizing, T-cells. In this context, embodiments of the presentinvention provide methods for generating large numbers (or substantiallypure sub-populations) of activated T cells that express certain markersand/or cell-surface receptors or produce certain cytokines that areoptimal for T cell-mediated immune responses. Such manipulated T cellsmay be used in the treatment and prevention of many diseases, such ascancer, infectious diseases, autoimmune diseases, allergies, immunedysfunction related to aging, or any other disease state where T cellsare desired for treatment. Further embodiments described herein relateto methods and compositions for the effective therapy of any theaforementioned diseases by utilizing T-cells with optimal reactivity,which cells are selected or screened using the compositions and/ormethods of the instant invention. The compositions and methods of thepresent invention are more effective over existing compositions andmethods not only with respect to the ability to generate larger numberof activated T-cells but also with regard to the significantly improvedeffectiveness of such T-cells in the in vivo setting. Accordingly, thecompositions and methods of the instant invention are useful for thegeneration of highly desirable human T lymphocytes for engraftment,autologous transfers, and for therapeutic applications.

Accordingly, in one embodiment, the instant invention provides antigenpresenting cell-mimetic scaffolds (APC-MS), comprising a base layercomprising high surface area mesoporous silica micro-rods (MSR); acontinuous, fluid supported lipid bilayer (SLB) layered on the MSR baselayer; a plurality of T-cell activating molecules and T-cellco-stimulatory molecules adsorbed onto the scaffold; and a plurality ofT-cell homeostatic agents adsorbed onto the scaffold.

In one embodiment, the present invention provides antigen presentingcell-mimetic scaffolds (APC-MS) that sequester T-cells selected from thegroup consisting of natural killer (NK) cells, CD3+ T-cells, CD4+T-cells, CD8+ T-cells, and regulatory T-cells (Tregs), or a combinationthereof.

In one embodiment, the present invention provides antigen presentingcell-mimetic scaffolds (APC-MS) containing the plurality of T-cellhomeostatic agents which are adsorbed onto the SLB layer.

In one embodiment, the present invention provides antigen presentingcell-mimetic scaffolds (APC-MS) containing the plurality of T-cellhomeostatic agents which are adsorbed onto the MSR layer.

In one embodiment, the present invention provides antigen presentingcell-mimetic scaffolds (APC-MS) containing the plurality of T-cellhomeostatic agents which are released from the scaffold in acontrolled-release manner. In some embodiments, the T-cell homeostaticagent is released from the scaffold in a controlled release manner overa period of 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 16days, 17 days, 18 days, 19 days, 20 days, 21 days, 22 days, 23 days, 24days, 25 days, 30 days, 35 days, 40 days, 45 days, 50 days, 60 days, 1month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8months, 9 months, or more.

In one embodiment, the present invention provides antigen presentingcell-mimetic scaffolds (APC-MS) containing the plurality of T-cellhomeostatic agents which are released from the scaffold in a sustainedmanner for up to 15 days. In some embodiments, the T-cell homeostaticagent is released from the scaffold in a sustained manner for up to 1day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10days, 11 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days, 18days, 19 days, 20 days, 21 days, 22 days, 23 days, 24 days, 25 days, 30days, 35 days, 40 days, 45 days, 50 days, 60 days, 1 month, 2 months, 3months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, ormore. In some embodiments, the T-cell homeostatic agent is released fromthe scaffold in a sustained manner for at least 30 days. In someembodiments, the T-cell homeostatic agent is released from the scaffoldin a sustained manner for at least 1 day, 2 days, 3 days, 4 days, 5days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13days, 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days, 21days, 22 days, 23 days, 24 days, 25 days, 30 days, 35 days, 40 days, 45days, 50 days, 60 days, 1 month, 2 months, 3 months, 4 months, 5 months,6 months, 7 months, 8 months, 9 months, or more.

In one embodiment, the present invention provides antigen presentingcell-mimetic scaffolds (APC-MS) containing the plurality of T-cellhomeostatic agents which are selected from the group consisting of IL-1,IL-2, IL-4, IL-5, IL-7, IL-10, IL-12, IL-15, IL-17, IL-21, andtransforming growth factor beta (TGF-β), or an agonist thereof, amimetic thereof, a variant thereof, a functional fragment thereof, or acombination thereof.

In one embodiment, the present invention provides antigen presentingcell-mimetic scaffolds (APC-MS) containing a plurality of the T-cellhomeostatic agents which are IL-2, an agonist thereof, a mimeticthereof, a variant thereof, a functional fragment thereof, or acombination thereof with a second homeostatic agent selected from thegroup consisting of IL-7, IL-21, IL-15, and IL-15 superagonist. In oneembodiment, the T-cell homeostatic agent may be selected from the groupconsisting of an N-terminal IL-2 fragment comprising the first 30 aminoacids of IL-2 (p1-30), an IL-2 superkine peptide, and an IL-2 partialagonist peptide, or a combination thereof.

In another embodiment, the present invention relates to antigenpresenting cell-mimetic scaffolds (APC-MS) containing a plurality ofactivating and co-stimulatory molecules, wherein the T-cell activatingmolecules and the T-cell co-stimulatory molecules are each,independently, adsorbed onto the fluid supported lipid bilayer (SLB). Inone embodiment, the T-cell activating molecules and the T-cellco-stimulatory molecules may be adsorbed via affinity pairing orchemical coupling. In some embodiments, the chemical coupling comprisesa click chemistry reagent (e.g., DBCO or azide). In one embodiment, theT-cell activating molecules and the T-cell co-stimulatory molecules maybe adsorbed via affinity pairing comprising a biotin-streptavidin pair,an antibody-antigen pair, an antibody-hapten pair, an aptamer affinitypair, a capture protein pair, an Fc receptor-IgG pair, a metal-chelatinglipid pair, a metal-chelating lipid-histidine (HIS)-tagged protein pair,or a combination thereof. In one embodiment, the T-cell activatingmolecules and the T-cell co-stimulatory molecules may be adsorbed viachemical coupling comprising azide-alkyne chemical (AAC) reaction,dibenzo-cyclooctyne ligation (DCL), or tetrazine-alkene ligation (TAL).

In another embodiment, the present invention relates to antigenpresenting cell-mimetic scaffolds (APC-MS) containing a plurality ofactivating and co-stimulatory molecules, wherein the T-cell activatingmolecules and the T-cell co-stimulatory molecules are each,independently, coated onto the fluid supported lipid bilayer (SLB).Alternately, in another embodiment, the present invention relates toantigen presenting cell-mimetic scaffolds (APC-MS) containing aplurality of activating and co-stimulatory molecules, wherein the T-cellactivating molecules and the T-cell co-stimulatory molecules are each,independently, partly embedded onto the fluid supported lipid bilayer(SLB).

In another embodiment, the present invention relates to antigenpresenting cell-mimetic scaffolds (APC-MS) containing a plurality ofactivating and co-stimulatory molecules, wherein the T-cell activatingmolecules and the T-cell co-stimulatory molecules are each,independently, adsorbed onto the mesoporous silica micro-rods (MSR).

In another embodiment, the present invention relates to antigenpresenting cell-mimetic scaffolds (APC-MS) containing a plurality ofactivating and co-stimulatory molecules, wherein the T-cell activatingmolecules and the T-cell co-stimulatory molecules are each,independently, antibody molecules or antigen-binding fragments thereof.

In another embodiment, the present invention relates to antigenpresenting cell-mimetic scaffolds (APC-MS) containing a plurality ofactivating and co-stimulatory molecules, wherein the T-cell activatingmolecules are selected from the group consisting of an anti-CD3 antibodyor an antigen-binding fragment thereof, an anti-CD2 antibody or anantigen-binding fragment thereof, an anti-CD47 antibody or anantigen-binding fragment thereof, anti-macrophage scavenger receptor(MSR1) antibody or an antigen-binding fragment thereof, an anti-T-cellreceptor (TCR) antibody or an antigen-binding fragment thereof, a majorhistocompatibility complex (MHC) molecule or a multimer thereof loadedwith an MHC peptide, and an MHC-immunoglobulin (Ig) conjugate or amultimer thereof, or a combination thereof.

In another embodiment, the present invention relates to antigenpresenting cell-mimetic scaffolds (APC-MS) containing a plurality ofactivating and co-stimulatory molecules, wherein the T-cellco-stimulatory molecules are antibodies, or an antigen-binding fragmentsthereof, which specifically bind to a co-stimulatory antigen selectedfrom the group consisting of CD28, 4.1BB (CD137), OX40 (CD134), CD27(TNFRSF7), GITR (CD357), CD30 (TNFRSF8), HVEM (CD270), LTβR (TNFRSF3),DR3 (TNFRSF25), ICOS (CD278), CD226 (DNAM1), CRTAM (CD355), TIM1(HAVCR1, KIM1), CD2 (LFA2, OX34), SLAM (CD150, SLAMF1), 2B4 (CD244,SLAMF4), Ly108 (NTBA, CD352, SLAMF6), CD84 (SLAMF5), Ly9 (CD229,SLAMF3), CD279 (PD1) and CRACC (CD319, BLAME).

In another embodiment, the present invention relates to antigenpresenting cell-mimetic scaffolds (APC-MS) containing a plurality ofactivating and co-stimulatory molecules, wherein the T-cell activatingmolecules and T-cell co-stimulatory molecules comprise bispecificantibodies or antigen binding fragments thereof.

In another embodiment, the present invention relates to antigenpresenting cell-mimetic scaffolds (APC-MS) containing a plurality ofactivating and co-stimulatory molecules, wherein the T-cell activatingmolecules and T-cell co-stimulatory molecules comprise a pair selectedfrom the group consisting of CD3/CD28, CD3/ICOS optionally together withCD28, CD3/CD27 optionally together with CD28, and CD3/CD137 optionallytogether with CD28, or a combination thereof.

In another embodiment, the present invention relates to antigenpresenting cell-mimetic scaffolds (APC-MS) which further comprise animmunoglobulin molecule that binds specifically to an Fc-fusion protein.

In another embodiment, the present invention relates to antigenpresenting cell-mimetic scaffolds (APC-MS) which further comprise arecruitment compound selected from the group consisting of granulocytemacrophage-colony stimulating factor (GM-CSF), chemokine (C-C motif)ligand 21 (CCL-21), chemokine (C-C motif) ligand 19 (CCL-19), a C-X-Cmotif chemokine ligand 12 (CXCL12), Interferon gamma (IFNγ), or aFMS-like tyrosine kinase 3 (Flt-3) ligand, or an agonist thereof, amimetic thereof, a variant thereof, a functional fragment thereof, or acombination thereof. In one embodiment, the scaffolds further comprise arecruitment compound which is granulocyte macrophage colony stimulatingfactor (GM-CSF), or an agonist thereof, a mimetic thereof, a variantthereof, or a functional fragment thereof.

In another embodiment, the present invention relates to antigenpresenting cell-mimetic scaffolds (APC-MS) which further comprise anantigen. In one embodiment, the antigen comprises a tumor antigen. Stillfurther under this embodiment, the tumor antigen is selected from thegroup consisting of MAGE-1, MAGE-2, MAGE-3, CEA, Tyrosinase, midkin,BAGE, CASP-8, β-catenin, β-catenin, γ-catenin, CA-125, CDK-1, CDK4,ESO-1, gp75, gp100, MART-1, MUC-1, MUM-1, p53, PAP, PSA, PSMA, ras,trp-1, HER-2, TRP-1, TRP-2, IL13Ralpha, IL13Ralpha2, AIM-2, AIM-3,NY-ESO-1, C9orf 112, SART1, SART2, SART3, BRAP, RTN4, GLEA2, TNKS2,KIAA0376, ING4, HSPH1, C13orf24, RBPSUH, C6orf153, NKTR, NSEP1, U2AF1L,CYNL2, TPR, SOX2, GOLGA, BMI1, COX-2, EGFRvIII, EZH2, LICAM, Livin,Livinβ, MRP-3, Nestin, OLIG2, ART1, ART4, B-cyclin, Gli1, Cav-1,cathepsin B, CD74, E-cadherin, EphA2/Eck, Fra-1/Fosl 1, GAGE-1,Ganglioside/GD2, GnT-V, β1,6-N, Ki67, Ku70/80, PROX1, PSCA, SOX10,SOX11, Survivin, UPAR, WT-1, Dipeptidyl peptidase IV (DPPIV), adenosinedeaminase-binding protein (AD Abp), cyclophilin b, Colorectal associatedantigen (CRC)-C017-1A/GA733, T-cell receptor/CD3-zeta chain, GAGE-familyof tumor antigens, RAGE, LAGE-I, NAG, GnT-V, RCAS1, α-fetoprotein,pl20ctn, Pmel117, PRAME, brain glycogen phosphorylase, SSX-I, SSX-2(HOM-MEL-40), SSX-I, SSX-4, SSX-5, SCP-I, CT-7, cdc27, adenomatouspolyposis coli protein (APC), fodrin, PlA, Connexin 37, Ig-idiotype,pl5, GM2, GD2 gangliosides, Smad family of tumor antigens, Imp-1,EBV-encoded nuclear antigen (EBNA)-I, UL16-binding protein-liketranscript 1 (Mult1), RAE-1 proteins, H60, MICA, MICB, c-erbB-2, aneoantigen identified in a patient specific manner, or an immunogenicpeptide thereof, or a combination thereof.

In a related embodiment, the present invention relates to antigenpresenting cell-mimetic scaffolds (APC-MS), comprising a base layercomprising high surface area mesoporous silica micro-rods (MSR); acontinuous, fluid supported lipid bilayer (SLB) layered on the MSR baselayer; a plurality of T-cell activating molecules and T-cellco-stimulatory molecules adsorbed onto the scaffold; and a plurality ofT-cell homeostatic agents adsorbed onto the scaffold, wherein the weightratio of the supported lipid bilayer (SLB) to the mesoporous silicamicro-rods (MSR) is between about 10:1 and about 1:20. In oneembodiment, the weight ratio reflects the ratio of SLB to MSR prior toloading. In another embodiment, the weight ratio is adjusted to achievethe desired scaffold composition. In one embodiment, the weight ratio ofthe SLB to the MSR may be between about 9:1 and about 1:15, betweenabout 5:1 and about 1:10, between about 3:1 and about 1:5, including allratios in between, e.g., about 3.1, about 2:1, about 1:1, about 1:2,about 1:3, about 1:4, about 1:5, about 1:6, about 1:7, about 1:8, about1:9, about 1:10.

In another embodiment, the present invention relates to antigenpresenting cell-mimetic scaffolds (APC-MS), comprising a base layercomprising high surface area mesoporous silica micro-rods (MSR); acontinuous, fluid supported lipid bilayer (SLB) layered on the MSR baselayer; a plurality of T-cell activating molecules and T-cellco-stimulatory molecules adsorbed onto the scaffold; and a plurality ofT-cell homeostatic agents adsorbed onto the scaffold, wherein thecontinuous, fluid supported lipid bilayer (SLB) comprises a lipidcomprising 14 to 23 carbon atoms.

In one embodiment, the lipid is phosphatidylethanolamine (PE),phosphatidylcholine (PC), phosphatidic acid (PA), phosphatidylserine(PS), or phosphoinositide, or a derivative thereof. In one embodiment,the APC-MS comprises fluid supported lipid bilayer (SLB) comprises alipid which is selected from the group consisting ofdimyristoylphosphatidylcholine (DMPC), dipalmitoylphosphatidylcholine(DPPC), distearoylphosphatidylcholine (DSPC),palmitoyl-oleoylphosphatidylcholine (POPC), dioleoylphosphatidylcholine(DOPC), dioleoylphosphatidylethanolamine (DOPE),dimyristoylphosphatidylethanolamine (DMPE), anddipalmitoylphosphatidylethanolamine (DPPE) or a combination thereof. Insome embodiments, the lipid bilayer comprises a lipid composition thatmimics the lipid composition of a mammalian cell membrane (e.g., a humancell plasma membrane). The lipid composition of many mammalian cellmembranes have been characterized and are readily ascertainable by oneof skill in the art (see, e.g., Essaid et al. Biochim. Biophys. Acta1858(11): 2725-36 (2016), the entire contents of which are incorporatedherein by reference). In some embodiments, the lipid bilayer comprisescholesterol. In some embodiments, the lipid bilayer comprises asphingolipid. In some embodiments, the lipid bilayer comprises aphospholipid. In some embodiments, the lipid is aphosphatidylethanolamine, a phosphatidylcholine, a phosphatidylserine, aphosphoinositide a phosphosphingolipid with saturated or unsaturatedtails comprising 6-20 carbons, or a combination thereof.

In another embodiment, the present invention relates to antigenpresenting cell-mimetic scaffolds (APC-MS), comprising a base layercomprising high surface area mesoporous silica micro-rods (MSR); acontinuous, fluid supported lipid bilayer (SLB) layered on the MSR baselayer; a plurality of T-cell activating molecules and T-cellco-stimulatory molecules adsorbed onto the scaffold; and a plurality ofT-cell homeostatic agents adsorbed onto the scaffold, wherein themesoporous silica microrod-lipid bilayer (MSR-SLB) scaffold retains acontinuous, fluid architecture for at least 14 days. In someembodiments, the MSR-SLB scaffold retains a continuous, fluidarchitecture for 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days,8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 16days, 17 days, 18 days, 19 days, 20 days, 21 days, 25 days, 30 days, 35days, 40 days, 50 days, or more. In some embodiments, the MSR of theMSR-SLB scaffold degrade in about 1 day, 2 days, 3 days, 4 days, 5 days,6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14days, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days, 21 days, 25days, 30 days, 35 days, 40 days, 50 days, or more. In some embodiments,the lipid bilayer of the MSR-SLB scaffold degrades in about 1 day, 2days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days,11 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days, 18 days,19 days, 20 days, 21 days, 25 days, 30 days, 35 days, 40 days, 50 days,or more.

In another embodiment, the present invention relates to antigenpresenting cell-mimetic scaffolds (APC-MS), comprising a base layercomprising high surface area mesoporous silica micro-rods (MSR); acontinuous, fluid supported lipid bilayer (SLB) layered on the MSR baselayer; a plurality of T-cell activating molecules and T-cellco-stimulatory molecules adsorbed onto the scaffold; and a plurality ofT-cell homeostatic agents adsorbed onto the scaffold, wherein the dryweight ratio of the mesoporous silica micro-rods (MSR) to the T-cellactivating/co-stimulatory molecules is between about 1:1 to about 50:1.In one embodiment, the ratio of MSR to T-cell activating/co-stimulatorymolecules is reflective of the weight of the MSR to the weight of theantibodies which are used as T-cell activating/co-stimulatory molecules.In another embodiment, the MSR:antibody weight ratio is adjusted toachieve the desired scaffold composition. In one embodiment, the weightratio of the SLB to the antibody composition is between about 2:1 andabout 20:1, between about 3:1 and about 10:1, between about 4:1 andabout 8:1, including all ratios in between, e.g., about 1:1, about 2:1,about 3:1, about 4:1, about 5:1, about 6:1, about 7:1, about 8:1, about9:1, about 10:1, about 15:1, about 20:1, about 25:1, about 30:1, about40:1.

In another embodiment, the present invention relates to antigenpresenting cell-mimetic scaffolds (APC-MS), comprising a base layercomprising high surface area mesoporous silica micro-rods (MSR); acontinuous, fluid supported lipid bilayer (SLB) layered on the MSR baselayer; a plurality of T-cell activating molecules and T-cellco-stimulatory molecules adsorbed onto the scaffold; and a plurality ofT-cell homeostatic agents adsorbed onto the scaffold, wherein thescaffolds are stacked to selectively permit infiltration of T-cells intothe mesoporous silica micro-rods (MSR). In one embodiment, the instantinvention further provides APC-MS wherein the T-cell activating and/orco-stimulatory molecules are present on the scaffolds at a concentrationsufficient to permit in situ manipulation of T-cells.

In another aspect, the present invention relates to pharmaceuticalcompositions comprising antigen presenting cell-mimetic scaffolds(APC-MS) comprising a base layer comprising high surface area mesoporoussilica micro-rods (MSR); a continuous, fluid supported lipid bilayer(SLB) layered on the MSR base layer; a plurality of T-cell activatingmolecules and T-cell co-stimulatory molecules adsorbed onto thescaffold; and a plurality of T-cell homeostatic agents adsorbed onto thescaffold; and a pharmaceutically acceptable carrier. In one embodiment,the instant invention further provides pharmaceutical compositions thatare formulated for intravenous administration, subcutaneousadministration, intraperitoneal administration, or intramuscularadministration.

In another aspect, the present invention relates to compositionscomprising antigen presenting cell-mimetic scaffolds (APC-MS) comprisinga base layer comprising high surface area mesoporous silica micro-rods(MSR); a continuous, fluid supported lipid bilayer (SLB) layered on theMSR base layer; a plurality of T-cell activating molecules and T-cellco-stimulatory molecules adsorbed onto the scaffold; and a plurality ofT-cell homeostatic agents adsorbed onto the scaffold; and T-cellsclustered therein. In one embodiment, the instant invention furtherprovides compositions that contain APC-MS and T-cells selected from thegroup consisting of natural killer (NK) cells, a CD3+ T-cells, CD4+T-cells, CD8+ T-cells, and regulatory T-cells (Tregs), or a combinationthereof.

Still further, embodiments of the instant invention relate to methods oftreating a disease in a subject in need thereof, comprising contacting asample comprising a T-cell population obtained from the subject with theantigen presenting cell-mimetic scaffold (APC-MS), thereby activating,co-stimulating and homeostatically maintaining the population ofT-cells; optionally expanding the population of T-cells; andadministering the activated, co-stimulated, maintained and optionallyexpanded T-cells into the subject, thereby treating the disease in thesubject. In one embodiment, the instant invention further providesmethods of treating a disease in a subject in need thereof, wherein themethod further comprises re-stimulating the population of T-cells priorto the administration step. In one embodiment, the method includesexpanding the population of T-cells after contacting with the scaffoldfor a period between 2 days to 5 days.

In another therapeutic embodiment, the instant invention relate tomethods of treating a disease in a subject in need thereof, comprisingcontacting a sample which is a blood sample, a bone marrow sample, alymphatic sample or a splenic sample comprising a T-cell populationobtained from the subject with the antigen presenting cell-mimeticscaffold (APC-MS), thereby activating, co-stimulating andhomeostatically maintaining the population of T-cells; optionallyexpanding the population of T-cells; and administering the activated,co-stimulated, maintained and optionally expanded T-cells into thesubject, thereby treating the disease in the subject. In one embodiment,the subject is a human subject. In one embodiment, the method providesfor the treatment of a cancer and the scaffold comprises at least onecytotoxic T-cell specific activating molecules and at least onecytotoxic T-cell specific co-stimulatory molecule.

In another therapeutic embodiment, the instant invention relate tomethods of treating a cancer in a subject in need thereof, comprisingcontacting a sample comprising a T-cell population obtained from thesubject with the antigen presenting cell-mimetic scaffold (APC-MS),thereby activating, co-stimulating and homeostatically maintaining thepopulation of T-cells; optionally expanding the population of T-cells;and administering the activated, co-stimulated, maintained andoptionally expanded T-cells into the subject, thereby treating thecancer in the subject. In one embodiment, the cancer is selected fromthe group consisting of head and neck cancer, breast cancer, pancreaticcancer, prostate cancer, renal cancer, esophageal cancer, bone cancer,testicular cancer, cervical cancer, gastrointestinal cancer,glioblastoma, leukemia, lymphoma, mantle cell lymphoma, pre-neoplasticlesions in the lung, colon cancer, melanoma, and bladder cancer. In oneembodiment, the method may further include sorting and optionallyenriching cytotoxic T-cells from the sample and/or the expanded cellpopulation.

In yet another therapeutic embodiment, the instant invention relate tomethods of treating an immunodeficiency disorder in a subject in needthereof, comprising contacting a sample comprising a T-cell populationobtained from the subject with the antigen presenting cell-mimeticscaffold (APC-MS), thereby activating, co-stimulating andhomeostatically maintaining the population of T-cells; optionallyexpanding the population of T-cells; and administering the activated,co-stimulated, maintained and optionally expanded T-cells into thesubject, thereby treating the immunodeficiency disorder in the subject.In one embodiment, the scaffold comprises at least one helper T-cell(Th) specific activating molecule and at least one helper T-cell (Th)specific co-stimulatory molecule. In one embodiment, the method may beused to treat an immunodeficiency disorder selected from the groupconsisting of primary immunodeficiency disorder and acquiredimmunodeficiency disorder. In one embodiment, the method may be used totreat acquired immunodeficiency syndrome (AIDS) or a hereditary disorderselected from the group consisting of DiGeorge syndrome (DGS),chromosomal breakage syndrome (CBS), ataxia telangiectasia (AT) andWiskott-Aldrich syndrome (WAS), or a combination thereof.

In another embodiment, the instant invention relates to methods oftreating a disease in a subject in need thereof, comprising contacting asample comprising a T-cell population obtained from the subject with theantigen presenting cell-mimetic scaffold (APC-MS), thereby activating,co-stimulating and homeostatically maintaining the population ofT-cells; optionally expanding the population of T-cells; further sortingand optionally enriching the T-cells from the sample and/or the expandedcell population; and administering the activated, co-stimulated,maintained and optionally expanded T-cells into the subject, therebytreating the disease in the subject. In one embodiment, the T-cells maybe selected from the group consisting of natural killer (NK) cells, aCD3+ T-cells, CD4+ T-cells, CD8+ T-cells, and regulatory T-cells(Tregs), or a combination thereof.

In another embodiment, the instant invention relates to methods oftreating an autoimmune disorder in a subject in need thereof, comprisingcontacting a sample comprising a T-cell population obtained from thesubject with the antigen presenting cell-mimetic scaffold (APC-MS),thereby activating, co-stimulating and homeostatically maintaining thepopulation of T-cells; optionally expanding the population of T-cells;further optionally sorting and enriching the T-cells from the sampleand/or the expanded cell population; and administering the activated,co-stimulated, maintained and optionally expanded T-cells into thesubject, thereby treating the autoimmune disorder in the subject.

In another embodiment, the instant invention relates to methods oftreating a disease in a subject in need thereof, comprising contacting asample comprising a T-cell population obtained from the subject with theantigen presenting cell-mimetic scaffold (APC-MS), thereby activating,co-stimulating and homeostatically maintaining the population ofT-cells; optionally expanding the population of T-cells; furtheroptionally sorting and enriching the T-cells from the sample and/or theexpanded cell population; and subcutaneously or intravenouslyadministering the activated, co-stimulated, maintained and optionallyexpanded T-cells into the subject, thereby treating the disease in thesubject. In one embodiment, the T-cells may be activated, co-stimulated,homeostatically maintained, and optionally expanded by contacting thesample with the scaffold for a period between about 1 day to about 20days.

In another embodiment, the instant invention relates to methods for themanipulation of T-cells, comprising contacting the antigen presentingcell-mimetic scaffold (APC-MS) with a subject's biological sample,thereby activating, co-stimulating, homeostatically maintaining andoptionally expanding a population of T-cells present within the sample,thereby manipulating the T-cells. In one embodiment, the manipulationmay include stimulation, activation, changes in viability, promotion ofgrowth, division, differentiation, expansion, proliferation, exhaustion,anergy, quiescence, apoptosis, death of T-cells. In one embodiment, themanipulation preferably includes promoting expansion or proliferation ofT-cells. In an additional embodiment, the manipulated T-cells may befurther transformed. In a specific embodiment, the T-cells may betransformed to express a chimeric antigen receptor (CAR). The CAR T-cellproduct may be further expanded by incubating with the antigenpresenting cell-mimetic scaffolds (APC-MS) containing an antigen whichis specific to the CAR T-cell. In certain embodiments, the CART-cell-specific antigen is selected from the group consisting of CD19,CD22, or a fragment thereof or a variant thereof. In some embodiments,the CAR T-cell-specific antigen is a tumor antigen. Tumor antigens arewell known in the art and include, for example, a glioma-associatedantigen, carcinoembryonic antigen (CEA), β-human chorionic gonadotropin,alphafetoprotein (AFP), lectin-reactive AFP, thyroglobulin, RAGE-1,MN-CA IX, human telomerase reverse transcriptase, RU1, RU2 (AS),intestinal carboxyl esterase, mut hsp70-2, M-CSF, prostase,prostate-specific antigen (PSA), PAP, NY-ESO-1, LAGE-la, p53, prostein,PSMA, Her2/neu, survivin and telomerase, prostate-carcinoma tumorantigen-1 (PCTA-1), MAGE, ELF2M, neutrophil elastase, ephrinB2, CD22,insulin growth factor (IGF)-I, IGF-II, IGF-I receptor and mesothelin. Insome embodiments, a CAR T-cell product may be expanded polyclonallypost-production to generate a larger population of CAR T-cells.

In another embodiment, the instant invention relates to methods for themanipulation of T-cells, comprising contacting the antigen presentingcell-mimetic scaffold (APC-MS), wherein the method confers increasedexpansion of the population of T-cells after about 1 week of contactwith the scaffold compared to a control scaffold comprising the baselayer comprising high surface area mesoporous silica micro-rods (MSR)and the continuous, fluid supported lipid bilayer (SLB) but notcontaining the T-cell activating molecules and the T-cell co-stimulatorymolecules. In one embodiment, the method confers about a 50-fold to800-fold increase in the expansion of the population of T-cells afterabout 1 week of contact with the scaffold compared to a control scaffoldcomprising the base layer comprising high surface area mesoporous silicamicro-rods (MSR) and the continuous, fluid supported lipid bilayer (SLB)but not containing the T-cell activating molecules and the T-cellco-stimulatory molecules.

In another embodiment, the instant invention relates to methods for themanipulation of T-cells, comprising contacting the antigen presentingcell-mimetic scaffold (APC-MS), wherein the method confers increasedexpansion of the population of T-cells after about 1 week of contactwith the scaffold compared to a superparamagnetic spherical polymerparticle (DYNABEAD) comprising the T-cell activating molecules and theT-cell co-stimulatory molecules. In one embodiment, the method confersabout a 5-fold to 20-fold increase in the expansion of the population ofT-cells after about 1 week of contact with the scaffold compared to asuperparamagnetic spherical polymer particle (DYNABEAD) comprising theT-cell activating molecules and the T-cell co-stimulatory molecules.

In another embodiment, the instant invention relates to methods forimproving the metabolic activity of T-cells, comprising contacting theantigen presenting cell-mimetic scaffold (APC-MS) with a subject'sbiological sample, thereby activating, co-stimulating, homeostaticallymaintaining and optionally expanding a population of T-cells presentwithin the sample, thereby improving the metabolic activity of T-cells.In one embodiment, the method confers improved metabolic activity of thepopulation of T-cells after about 1 week of contact with the scaffoldcompared to a control scaffold comprising the base layer comprising highsurface area mesoporous silica micro-rods (MSR) and the continuous,fluid supported lipid bilayer (SLB) but not containing the T-cellactivating molecules and the T-cell co-stimulatory molecules. In oneembodiment, the method confers about a 5-fold to 20-fold improvedmetabolic activity of the population of T-cells after about 1 week ofcontact with the scaffold compared to a control scaffold comprising thebase layer comprising high surface area mesoporous silica micro-rods(MSR) and the continuous, fluid supported lipid bilayer (SLB) but notcontaining the T-cell activating molecules and the T-cell co-stimulatorymolecules. In one embodiment, the method confers improved metabolicactivity of the population of T-cells after about 1 week of contact withthe scaffold compared to a superparamagnetic spherical polymer particle(DYNABEAD) comprising the T-cell activating molecules and the T-cellco-stimulatory molecules.

In one embodiment, the method further confers about a 1-fold to 10-foldincrease in the expansion of the population of T-cells after about 1week of contact with the scaffold compared to a superparamagneticspherical polymer particle (DYNABEAD) comprising the T-cell activatingmolecules and the T-cell co-stimulatory molecules.

In another embodiment, the instant invention relates to methods forscreening metabolically active T-cells, comprising contacting theantigen presenting cell-mimetic scaffold (APC-MS) with a subject'sbiological sample, thereby activating, co-stimulating, homeostaticallymaintaining and optionally expanding a population of T-cells presentwithin the sample; identifying metabolically active cells in thepopulation of activated, co-stimulated, homeostatically maintained andoptionally expanded T-cells; thereby screening metabolically-activeT-cells. In one embodiment, the expanded T-cells are metabolicallyactive for at least about 7 days post-contact with the scaffold. In oneembodiment, the expanded T-cells form aggregates for at least about 7days post-contact with the scaffold.

Yet in another embodiment, the instant invention relates to methods forgenerating a polyclonal population of T-cells, comprising contacting theantigen presenting cell-mimetic scaffold (APC-MS) with a subject'sbiological sample, thereby activating, co-stimulating, homeostaticallymaintaining and optionally expanding a population of T-cells presentwithin the sample; identifying a specific population of T-cells from theexpanded population of T-cells based on the expression of a plurality ofmarkers in the expanded T-cells; optionally isolating or purifying theidentified population of T-cells, thereby generating a polyclonalpopulation of T-cells. In one embodiment, the method may be adapted forthe generation of a polyclonal population of CD4+ cells or CD8+ cells.In a related embodiment, the method may be adapted for the generation ofa polyclonal population of CD4+/FOXP3+ T-cells. Still further, themethod may be adapted for the generation of a polyclonal population ofCD44+/CD62L− T-cells (effector memory and/or effector T-cells). Inanother embodiment, the method may be adapted for the generation of apolyclonal population of CD8+/CD69+ T-cells (activated T-cells). Inanother embodiment, the method may be adapted for the generation of apolyclonal population of granzyme B+ CD8+ T-cells (cytotoxin-secretingT-cells). In yet another embodiment, the method may be adapted for thegeneration of a polyclonal population of IFNγ+ T-cells (activatorcytokine-secreting T-cells). In yet another embodiment, the method maybe adapted for the generation of a polyclonal population of CD62L+/CCR7+T-cells (memory T-cells).

In another embodiment, the instant invention relates to methods forgenerating a polyclonal sub-population of T-cells, comprising contactingthe antigen presenting cell-mimetic scaffold (APC-MS) with a subject'sbiological sample, thereby activating, co-stimulating, homeostaticallymaintaining and optionally expanding a population of T-cells presentwithin the sample; identifying a specific population of exhaustedT-cells from the expanded population of T-cells based on the expressionof a plurality of markers in the expanded T-cells; optionally removingthe identified population of T-cells, thereby generating a polyclonalsub-population of T-cells. In one embodiment, the exhausted T-cells areidentified or isolated based on cell-surface expression of CD8+/PD-1+.In another embodiments, the exhausted T-cells are identified or isolatedbased on cell-surface expression of LAG3+/TIM3+.

In another embodiment, the instant invention relates to methods formanipulation of T-cells ex vivo, comprising contacting the antigenpresenting cell-mimetic scaffold (APC-MS) with a subject's biologicalsample ex vivo, thereby activating, co-stimulating, homeostaticallymaintaining and optionally expanding a population of T-cells presentwithin the sample, thereby manipulating the T-cells ex vivo. In oneembodiment, the sample is contacted with the scaffold for a period fromabout 1 day to about 20 days. In one embodiment, the method may involvedetecting the production of one or more cytokines or cytotoxins producedby the manipulated T-cells. In one embodiment, the method involvesfurther detecting the production of a cytokine selected from the groupconsisting of interferon gamma (IFNγ), tissue necrosis factor alpha(TNFα), IL-2, IL-1, IL-4, IL-5, IL-10, and IL-13, IL-17 or a combinationthereof by the manipulated T-cells.

In a related embodiment, the instant invention relates to methods forthe manipulation of T-cells ex vivo in accordance with the foregoingmethods, wherein the manipulated T-cells are T-helper 1 (Th1) cells andthe method comprises detecting the production of a cytokine selectedfrom the group consisting of IL-2, interferon gamma (IFNγ) and tissuenecrosis factor alpha (TNFα), or a combination thereof. Alternately, ina related embodiment, the instant invention relates to methods for themanipulation of T-cells ex vivo in accordance with the foregoingmethods, wherein the manipulated T-cells are T-helper 2 (Th2) cells andthe method comprises detecting the production of a cytokine selectedfrom the group consisting of IL-4, IL-5, IL-10 and IL-13, or acombination thereof. Still further in a related embodiment, the instantinvention relates to methods for the manipulation of T-cells ex vivo inaccordance with the foregoing methods, wherein the manipulated T-cellsare cytotoxic T (Tc) cells and the method comprises detecting theproduction of a cytokine selected from the group consisting ofinterferon gamma (IFNγ) and lymphotoxin alpha (LTα/TNFβ), or acombination thereof. In one embodiment, the manipulated T-cells arecytotoxic T (Tc) cells and the method comprises detecting the secretionof a cytotoxin selected from the group consisting of a granzyme or aperforin, or a combination thereof.

In a related embodiment, the instant invention relates to methods forthe manipulation of T-cells ex vivo in accordance with the foregoingmethods, wherein the method further comprising detecting the expressionof a cell-surface marker in the manipulated T-cells. In one embodiment,the cell surface marker is selected from the group consisting of CD69,CD4, CD8, CD25, CD62L, FOXP3, HLA-DR, CD28, and CD134, or a combinationthereof. Alternately or additionally, in one embodiment, thecell-surface marker is a non-T-cell marker selected from the groupconsisting of CD36, CD40, and CD44, or a combination thereof.

In another related embodiment, the instant invention relates to methodsfor the manipulation of T-cells ex vivo in accordance with the foregoingmethods, wherein the subject is a human subject.

In another related embodiment, the instant invention relates to methodsfor the manipulation of T-cells in vivo in accordance with the foregoingmethods, wherein the scaffold is administered to the subject to permitthe biological sample comprising T-cells to come into contact with thescaffold in vivo. In one embodiment, the scaffold may be maintained inthe subject for a period between about 3 days to about 15 days,preferably for a period between about 7 days to about 11 days. In someembodiments, the scaffold may be maintained in the subject for a periodof at least 1 day, at least 2 days, at least 3 days, at least 4 days, atleast 5 days, at least 6 days, at least 7 days, at least 8 days, atleast 9 days, at least 10 days, at least 11 days, at least 12 days, atleast 13 days, at least 14 days, at least 15 days, at least 16 days, atleast 17 days, at least 18 days, at least 19 days, at least 20 days, atleast 21 days, at least 25 days, at least 30 days, at least 35 days, atleast 40 days, at least 50 days, or more.

In yet another embodiment, the instant invention relates to methods formaking the antigen presenting cell-mimetic scaffold (APC-MS), comprising(a) providing a base layer comprising high surface area mesoporoussilica micro-rods (MSR); (b) optionally loading the T-cell homeostaticagents on the MSR; (c) layering a continuous, fluid supported lipidbilayer (SLB) on the base layer comprising the MSRs, thereby generatingan MSR-SLB scaffold; (d) loading the T-cell homeostatic agents on theMSR-SLB scaffold if step (b) is not carried out; (e) optionally blockingone or more non-specific integration sites in the MSR-SLB scaffold witha blocker; and (f) loading the T-cell activating molecules and theT-cell co-stimulatory molecules onto the MSR-SLB scaffold, therebymaking the APC-MS. In one embodiment, the methods may further involveassembling a plurality of scaffolds to generate stacks with sufficientporosity to permit infiltration of T cells. In one embodiment, themethod may include loading at least one additional agent selected fromthe group consisting of a growth factor, a cytokine, an interleukin, anadhesion signaling molecule, an integrin signaling molecule, or afragment thereof or a combination thereof.

Other features and advantages of the invention will be apparent from thefollowing detailed description and the drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows phase-contrast and fluorescence microscope images of lipidsin association with mesoporous silica microrods (MSRs). The top panelshows merged pictures of the lipids and mesoporous silica microrods at alipid:MSR ratio of 1:20 (Scale=200 m). The middle panel shows mergedpictures of the lipids and mesoporous silica microrods at a lipid:MSRratio of 1:4 (Scale=200 m). The bottom panel shows a mergedphase-contrast microscope image of lipids in association with MSRs at ahigher magnification (Scale=20 m).

FIGS. 2A, 2B, 2C, and 2D show that the assembly and the characteristicsof the antigen-presenting cell-mimetic scaffolds (APC-MS) is dependenton the type of lipid and the content of the lipid. FIG. 2A showschemical structures of various lipids. Abbreviations:DOPC-dioleoylphosphatidylcholine;POPC-palmitoyl-oleoylphosphatidylcholine; andDSPC-distearoylphosphatidylcholine. FIG. 2B shows the percentage oflipid that is retained in various compositions containing mesoporoussilica microrods (MSR) and fluid supported, lipid bilayer (SLB). In thisexperiment, a payload of 250 μg lipid was inputted into a 500 μg MSRcomposition.

FIG. 2C shows changes in relative florescence of various MSR-SLBcompositions containing DOPC, POPC or DSPC in phosphate-buffered saline(PBS) over a two-week (14-day) period at 37° C. FIG. 2D shows changes inrelative florescence of various MSR-SLB compositions containing DOPC,POPC or DSPC in complete Roswell Park Memorial Institute medium (cRPMI)over a two-week (14-day) period at 37° C.

FIG. 3 shows stability of various MSR-SLB compositions in PBS at day 0,day 3, day 7, and day 14, as analyzed with phase-contrast andfluorescence microscopy (lipid coating). The top panel shows thestability of DOPC in the MSR-SLB composition; the middle panel shows thestability of POPC in the MSR-SLB composition; and the bottom panel showsthe stability of DSPC in the MSR-SLB composition.

FIGS. 4A, 4B, 4C, 4D, and 4E show changes in the assembly and thecharacteristics of MSR-SLB fluid structures over time. FIG. 4A showsphase-contrast and fluorescence microscope images of lipids inassociation with mesoporous silica microrods (MSRs) taken at highmagnification (scale=2 μM) prior to bleaching (pre), right afterbleaching (t=0) and 5 minutes post-bleaching (t=5 min) the lipidcomposition. FIG. 4B shows changes in fluorescence recovery afterphoto-bleaching (FRAP) with time. The fluorescence “source” is depictedin region (2), the fluorescence “sink” is depicted in region (3), andthe normalization point is indicated by region (1). The differentialdistribution was best seen at early time points after seeding andachieved an equilibrium at around 2 mins (120 s). FIG. 4C showssmooth-fitting curves depicting average changes in FRAP, as derived fromnormalized images, over time. FIGS. 4D and 4E show two sets of highresolution images of MSR-SLB fluid structures prior to bleaching (pre),right after bleaching (t=0) and 3 minutes post-bleaching (t=3 min) thelipid composition.

FIGS. 5A and 5B show structural and functional properties of MSR-SLBcompositions containing various moieties. Based on experiments using theB3Z reporter T-cell line, maximum functionality of the APC-MS scaffoldwas observed when all the individual components are present in thescaffold. FIG. 5A shows a schematic representation of the structure ofAPC-MS containing a lipid bilayer of POPC containing phycoerythrinbiotin (biotin PE), which is conjugated to a streptavidin molecule(e.g., a streptavidin dimer), which in turn is conjugated to abiotinylated antibody (e.g., a biotinylated anti-CD3 antibody or abiotinylated anti-CD28 antibody or another specific or non-specificantibody). FIG. 5B shows spectrophotometric analysis of B3Z reportercell β-galactosidase expression following treatment with combinations ofMPS (silica), POPC (lipid), MPS-POPC composite, biotinylated MPS-POPCcomposite (in the presence or absence of streptavidin) and the MPS-POPCcomposite together with the biotinylated antibody in the presence orabsence of phycoerythrin biotin (biotin PE) and/or streptavidin.Significant increase in absorbance is observed in MSR-SLB compositionscontaining all the individual components-phosphoethanolamine biotin(biotin PE) conjugated to a biotinylated antibody via a streptavidinlinker (dark bars; ** indicates statistical significance (p<0.001,analyzed using one-way ANOVA, followed by Tukey HSD post-hoc test; datarepresents mean±s.d. of three experimental replicates and arerepresentative of at least two independent experiments).

FIGS. 6A and 6B show controlled release of IL-2 from MSR-SLBcompositions containing IL-2. FIG. 6A shows an electron micrograph ofporous structure of MSR containing IL-2 (scale bar=100 nm). FIG. 6Bshows a plot of cumulative release of IL-2 levels over a 15-day period.

FIGS. 7A and 7B show confocal microscopy images showing infiltration ofT-cells (spheres) into the antigen presenting cell-mimetic scaffoldscontaining MSR-SLB composites. FIG. 7A shows cells that have beenstained with two different dyes. FIG. 7B shows cells that have beenstained with a single dye (indicating live cells).

FIG. 8 shows phase-contrast microscope and fluorescence images of lipidsin association with mesoporous silica microrods (MSRs) co-cultured withprimary T cells. It was observed that primary T cells tend to formcell/material clusters when T cell activating cues are attached to thesurface of the material. The bottom panel shows merged pictures of thelipids and mesoporous silica microrods in MSR-SLB composites containingconjugated antibodies, IL-2 or a combination of conjugated antibodiesand IL-2. The images on the right show MSR-SLB composites containingboth conjugated antibodies and IL-2 (Scale=20 m) at high magnification.

FIGS. 9A and 9B shows dose-response charts of antibody-induced changesin mouse splenic T cells. FIG. 9A shows polyclonal expansion of T-cellsafter a 3 day stimulation of T-cells with control scaffolds (mock; free;POPC lipid only; and a combination of POPC and IL-2) and experimentalscaffolds (containing a combination of POPC and IL-2, along withantibody). Three different doses of the antibody (MSR: antibody ratio of1:50, 1:25 and 1:10) were studied. FIG. 9B shows secretion of IFNγ aftera 3 day stimulation of T-cells with control scaffolds (mock; free; POPClipid only; and a combination of POPC and IL-2) and experimentalscaffolds (containing a combination of POPC and IL-2, along withantibody). Three different doses of the antibody (MSR: antibody ratio of1:50, 1:25 and 1:10) were studied.

FIGS. 10 and 11 show antigen-presenting cell-mimetic scaffolds (APC-MS)of the present invention promote rapid expansion of metabolically-activeT cells. FIG. 10 shows fold-expansion of primary T-cells upon incubationwith control (mock; free; SLB+IL-2; DYNABEAD+IL-2) or experimentalcompositions. Incubation of primary T-cells with the composition of theinstant invention significantly induced T-cell expansion (with orwithout re-stimulation) compared to mock compositions or compositionsfree of SLB. More importantly, compared to a composition of DYNABEADSand IL-2, incubation of primary T-cells with the scaffolds of theinvention resulted in a measurably stronger proliferation uponre-stimulation at day 7. FIG. 11 shows a bar-chart of cellular metabolicactivity of T-cells (as measured by relative fluorescence units (RFU) ofAlamar Blue reduction normalized to the cell number) that were incubatedwith the scaffolds of the instant invention loaded with IL-2(SLB/IL2/ABS) or DYNABEADS loaded with IL-2 (DYNABEADS-IL2).

FIGS. 12A and 12B show that the scaffolds of the invention (APC-MS)confer polyclonal expansion of splenic T cells (mouse) and facilitateformation of T cell aggregates. FIG. 12A shows photomicrographs (at 4×magnification) of aggregates of splenic T cells upon incubation withDYNABEADS or APC-MS at day 0, day 3, and day 7. FIG. 12B showsphotomicrographs (at 10× magnification) of aggregates of splenic T cellsupon incubation with DYNABEADS or APC-MS at day 0, day 3, and day 7.(White scale bars=100 μM).

FIGS. 13A and 13B show polyclonal expansion of mouse splenic T cellsupon incubation with APC-MS or DYNABEADS. FIG. 13A shows flow cytometric(FACS) scatter plots of T-cell population(s) at various time-points (t=0days, 5 days, 7 days, 11 days and 13 days) following incubation withAPC-MS or DYNABEADS (with re-stimulation or IL-2 treatment after 7 daysof incubation), wherein the values on the X-axis depict intensity ofCD8+ staining and the values on the Y-axis depict intensity of CD4+staining. Flow data were gated on Fluorescence Minus ONE (FMO) controlsfor each sample, at each timepoint. Data is representative of at leasttwo independent experiments. FIG. 13B is a line-graph showing changes inpercentage of CD4+ versus CD8+ T-cell sub-populations after incubationwith APC-MS (squares) or DYNABEADS (triangles) at various time-points(t=0 days, 5 days, 7 days, 11 days and 13 days). After 7-days ofincubation, the cells were divided into two sub-populations, wherein thefirst sub-population was re-stimulated (dashed line) and the secondsub-population was treated with IL-2 (solid line). APC-MS was used forrestimulation of APC-MS conditions, DYNABEADS were used to restimulateDYNABEADS conditions.

FIG. 14 shows measurement of polyclonal expansion of a subset of FoxP3+mouse splenic T cells upon incubation with APC-MS or DYNABEADS. Theresults are depicted in the form of flow cytometric (FACS) scatter plotsof T-cell population(s) at various time-points (t=0 days, 5 days, 7days, 11 days and 13 days) following incubation with APC-MS or DYNABEADS(with re-stimulation or IL-2 treatment after 7 days of incubation),wherein the values on the X-axis depict intensity of FoxP3+ staining andthe values on the Y-axis depict intensity of CD4+ staining. Arectangular gate was applied to count the number and/or proportion ofFoxP3+ cells in the various fractions. As shown, there was limited or noexpansion of FoxP3+ mouse splenic T cells with the particularformulation.

FIG. 15 shows polyclonal expansion of a subset of CD62L+ mouse splenic Tcells upon incubation with APC-MS or DYNABEADS. The results are depictedin the form of flow cytometric (FACS) scatter plots of T-cellpopulation(s) at various time-points (t=0 days, 5 days, 7 days, 11 daysand 13 days) following incubation with APC-MS or DYNABEADS (withre-stimulation or IL-2 treatment after 7 days of incubation), whereinthe values on the X-axis depict intensity of CD62L+ staining and thevalues on the Y-axis depict intensity of CD44+ staining. The CD62L+cells appear in the right hand (top and bottom right quadrants) of thescatter plots.

FIG. 16 shows polyclonal expansion of a subset of CD8+/CD69+ mousesplenic T cells upon incubation with APC-MS or DYNABEADS. The resultsare depicted in the form of flow cytometric (FACS) scatter plots ofT-cell population(s) at various time-points (t=0 days, 5 days, 7 days,11 days and 13 days) following incubation with APC-MS or DYNABEADS (withre-stimulation or IL-2 treatment after 7 days of incubation), whereinthe values on the X-axis depict intensity of CD8+ staining and thevalues on the Y-axis depict intensity of CD69+ staining. The CD8+/CD69+cells appear in the top right hand quadrant of the scatter plots.

FIG. 17 shows polyclonal expansion of a subset of CD8+/Granzyme B+ mousesplenic T cells upon incubation with APC-MS or DYNABEADS. The resultsare depicted in the form of flow cytometric (FACS) scatter plots ofT-cell population(s) at various time-points (t=0 days, 5 days, 7 days,11 days and 13 days) following incubation with APC-MS or DYNABEADS (withre-stimulation or IL-2 treatment after 7 days of incubation), whereinthe values on the X-axis depict intensity of CD8+ staining and thevalues on the Y-axis depict intensity of Granzyme B+ staining. TheCD8+/Granzyme B+ cells appear in the top right hand quadrant of thescatter plots.

FIG. 18 shows T-cell secretion of IFNγ (pg/cell) at various time-points(t=0 days, 5 days, 7 days, 11 days and 13 days) following incubationwith APC-MS (squares) or DYNABEADS (triangles). After 7-days ofincubation, the cells were divided into two sub-populations, wherein thefirst sub-population was re-stimulated (dashed line) and the secondsub-population was treated with IL-2 (solid line). Herein, APC-MS wasused in the re-stimulation of both APC-MS-incubated andDYNABEAD-incubated cell populations.

FIG. 19 shows levels of PD-1+ mouse splenic T cells upon incubation withAPC-MS or DYNABEADS. The results are depicted in the form of flowcytometric (FACS) scatter plots of T-cell population(s) at varioustime-points (t=0 days, 5 days, 7 days, 11 days and 13 days) followingincubation with APC-MS or DYNABEADS (with re-stimulation or IL-2treatment after 7 days of incubation), wherein the values on the X-axisdepict intensity of CD8+ staining and the values on the Y-axis depictintensity of PD-1+ staining (a potential marker of exhaustion).

FIGS. 20A and 20B show the effect of incubating human peripheral bloodT-cells with various compositions. FIG. 20A shows a line graph of thepolyclonal expansion of primary T cells that were incubated with controlscaffolds or experimental scaffolds at various time-points (t=0 days, 5days, 7 days, 11 days and 13 days). The control scaffolds include sham(“mock”; black line) compositions and compositions that are free of SLB(“free”; red line). The experimental scaffolds include (1) DYNABEADS(blue line) and (2) lipid bilayers (SLB) of the present invention (greenline). FIG. 20B shows a bar graph showing metabolic activity of primaryT cells (measured with standard Alamar Blue staining assay) that wereincubated with control scaffolds or experimental scaffolds at varioustime-points (t=0 days, 5 days, 7 days, 11 days and 13 days). The controlscaffolds include sham compositions (“mock”; “m”) and compositions thatare free of SLB (“free”; “f”). The experimental scaffolds include (1)DYNABEADS (“d”) and (2) lipid bilayers (SLB) of the present invention(“s”).

FIGS. 21A and 21B show the effect of incubating human peripheral bloodT-cells with various anti-CD3 antibodies. Human blood samples obtainedfrom subject 1 (FIG. 21A) and subject 2 (FIG. 21B) were incubated withcontrol scaffolds (“mock”) or experimental scaffolds containing thelisted anti-CD3 antibodies-muromonab (OKT3), an antibody recognizing17-19 kDa ε-chain of CD3 within the CD3 antigen/T cell antigen receptor(TCR) complex (HIT3a) and a monoclonal antibody recognizing a 20 kDasubunit of the TCR complex within CD3e (UCHT1). Three different dosageswere investigated—5 μg (top slides), 1 μg (bottom slide for subject 2)and 0.5 μg (bottom slide for subject 1). In each case, co-stimulationwas provided with anti-CD28 antibodies, wherein the ratio of anti-CD3antibody:anti-CD28 antibody was maintained at 1:1. Fold expansion of Tcells was measured at various time-points (t=0 days, 7 days, 11 days and13 days).

FIG. 22 shows polyclonal expansion of a human T cells upon incubationwith control scaffolds (“mock”) or experimental scaffolds containing thelisted anti-CD3 antibodies—OKT3, HIT3a, and UCHT1. The bottom panelsshow flow cytometric (FACS) scatter plots of T-cell population(s) atvarious time-points (t=8 days, 11 days and 14 days) following incubationwith APC-MS containing each of the anti-CD3 antibodies as a stimulatorymolecule and an anti-CD28 antibody as the co-stimulatory molecule. Thevalues on the X-axis of the scatter plots depict intensity of CD8+staining and the values on the Y-axis depict intensity of CD4+ staining.The plots are summarized in the line-graphs of the top panel, which showchanges in percentage of CD4+ versus CD8+ T-cell sub-populations afterincubation with APC-MS containing the aforementioned anti-CD3antibodies—OKT3 (circles), HIT3a (squares) and UCHT1 (triangles). Twodifferent antibody dosages were investigated—5 μg (1× dilution) and 0.5μg (1:10× dilution).

FIG. 23 shows CD62L and CCR7 expression on live T cells expanded for 14days using the APC-MS containing IL-2 and the aforementioned anti-CD3antibodies—OKT3 (left panels), HIT3a (middle panels) and UCHT1 (rightpanels) and anti-CD28 antibody at a 1:1 ratio, at 1× loadingconcentration (about 5 μg). The expression of CD62L and CCR7 in totallive cells is shown in the top panels and the expression of thesemarkers in gated CD8+ cells is shown in the bottom panels. A majority ofcells expanded with the APC-MS of the instant invention remainCD62L+CCR7+ after incubation for 14 days, which has been shown to beimportant for in vivo functionality in human patients. Additionally,APC-MS scaffolds containing OKT3 were particularly effective inexpanding and/or retaining CD62L+CCR7+ T-cells compared to scaffoldscontaining UCHT1 and/or HIT3a.

FIG. 24 outlines a representative scheme for making the scaffolds of theinstant invention.

FIGS. 25A and 25B depict the design of antigen-presenting cell-mimeticscaffolds (APC-MS). FIG. 25A depicts an exemplary process for preparingAPC-MS: 1) Mesoporous silica micro-rods (MSRs) are synthesized; 2) MSRsare adsorbed with IL-2; 3) IL-2-adsorbed MSRs are coated with liposomes,forming MSR-SLBs; 4) T cell activation cues are attached to the surfaceof MSR-SLBs; 5) MSR-SLBs are cultured with T cells; and 6) MSR-SLBssettle and stack to form a scaffold that is infiltrated by T cells.Scaffolds formed from MSR-SLBs that were loaded with IL-2 andsurface-functionalized with T cell activation cues are referred to asAPC-MS. FIG. 25B depicts exemplary structures and functions of distinctAPC-MS formulations. IL-2 is released from APC-MS over time, resultingin paracrine delivery of IL-2 to local T cells. Incorporation ofpredefined amounts of a biotinylated phospholipid into liposomeformulations enables the precise surface attachment of biotinylated Tcell activation cues via streptavidin-biotin interactions, mimicking thecell surface presentation of cues by natural APCs to T cells. Forpolyclonal T cell expansion, activating antibodies against CD3 (αCD3)and CD28 (αCD28) are attached (left). For antigen-specific T cellexpansion, peptide-loaded MHC (pMHC) and αCD28 are attached (right).

FIGS. 26A and 26B depicts the physical characterization of componentsused to assembly MSR-SLBs. FIG. 26A shows a representative brightfieldmicroscopy image of MSRs. Scale bar=100 μm. FIG. 26B depicts the sizedistribution of POPC liposomes as measured by dynamic light scattering(DLS). Data in FIG. 26B represents the mean size distribution of 3samples.

FIGS. 27A and 27B are microscopy images of lipid-coated MSRs. FIG. 27Ais a microscopy image showing the aggregation of MSRs at low lipid:MSR.Representative microscopy images of lipid-coated MSRs (lipid:MSR 1:20w/w) showing brightfield image of MSRs (left), fluorophore-taggedphospholipid (1 mol % of total lipid; middle), and co-localization ofMSRs and lipid (right). Scale bar=200 μm. FIG. 27B is a microscopy imageof lipid-coated MSRs (lipid:MSR 1:4 w/w) showing brightfield image ofMSRs (left), fluorophore-tagged phospholipid (1 mol % of total lipid;middle), and co-localization of MSRs and lipid (right). Scale bar=200μm.

FIGS. 28A-28E depict the assembly and characterization of APC-MS. FIG.28A depicts the retention of lipid coating (containing 1 mol %fluorophore-tagged lipid) on MSRs over time in either PBS or RPMI mediacontaining 10% serum (cRPMI), maintained at cell culture conditions.FIG. 28B is a representative overlaid fluorescence microscopy images oflipid-coated MSRs (MSRs, brightfield; lipid (1 mol % fluorophore-taggedlipid), green), maintained in cRPMI under standard cell cultureconditions, over time. Scale bar=100 μm. Data represents mean±s.d. ofthree experimental replicates and are representative of at least twoindependent experiments. FIG. 28C is a graph depicting thequantification of IL-2 released from MSR-SLBs (500 μg of MSRs) in vitroover time (data points) with one phase exponential fit (dashed line;R²=0.98). Data represents mean±s.d. of three experimental replicates andare representative of at least two independent experiments. FIG. 28D isa graph depicting the quantification of attachment of various inputs ofbiotinylated IgG onto MSRs coated with lipid formulations containing0.01 mol %, 0.1 mol %, or 1 mol % biotinylated lipid. Values above barsindicate concentration (μg) of IgG attached for each respectivecondition. Data represents mean±s.d. of four experimental replicates andare representative of at least two independent experiments. FIG. 28E isa SEM image showing close association of primary human T cells withAPC-MS. Scale bar=10 μm.

FIG. 29 shows the association of T cells with APC-MS. Representativemicroscopy images of MSR-SLBs either not presenting any surface cues(cue−), or surface-presenting αCD3 and αCD28 (cue+), at low (left) andhigh (right) magnification, cultured with primary mouse T cells for oneday. Cells and material are visible in brightfield images (top) andMSR-SLB lipid coatings are visible in the green channel (1 mol %fluorophore-tagged lipid; middle). Merged images are shown on thebottom. Low magnification scale bar=500 μm, high magnification scalebar=100 μm.

FIGS. 30A, 30B, 30C, 30D, 30E, 30F, and 30G show the polyclonalexpansion of primary mouse and human T cells. FIG. 30A arerepresentative brightfield microscopy images of primary mouse T cellscultured with DYNABEADS or APC-MS, at various timepoints, at lowmagnification (left) or high magnification with APC-MS (right). Scalebars=100 μm. FIG. 30B shows the expansion of primary mouse T cells thatwere either untreated (mock), or cultured with free cues (110 nM αCD3,110 nM αCD28, 1.3 μg/ml IL-2), commercial CD3/CD28 mouse T cellexpansion beads and exogenous IL-2 (DYNABEADS), IL-2-loaded MSR-SLBswithout T cell cues presented on the bilayer surface (MSR-SLB (cue-)),or APC-MS (loaded with αCD3, αCD28, IL-2). Curves for mock and free wereindistinguishable from the MSR-SLB (cue-) curve. FIG. 30C depicts thefrequencies of CD4+ and CD8+ cells among live single cells over time inAPC-MS or Dynabead cultures, measured using FACS. Data was analyzedusing two-way ANOVA, followed by Tukey HSD post-hoc test. FIG. 30D arerepresentative brightfield microscopy images of primary human T cellscultured with DYNABEADS or APC-MS formulations, at various timepoints.Scale bars=100 μm. (F1) APC-MS presenting αCD3and αCD28 saturating 1 mol% biotinylated lipid, input at 333 μg/ml of MSRs to initial culture,(F2) APC-MS presenting αCD3and αCD28 saturating 1 mol % biotinylatedlipid, input at 33 μg/ml of MSRs to initial culture, (F3) APC-MSpresenting αCD3and αCD28 saturating 0.1 mol % biotinylated lipid, inputat 333 μg/ml of MSRs to initial culture, and (F4) APC-MS presentingαCD3and αCD28 saturating 0.1 mol % biotinylated lipid, input at 33 μg/mlof MSRs to initial culture. FIG. 30E shows the expansion of primaryhuman T cells that were either untreated (mock), or cultured withcommercial CD3/CD28 human T cell expansion beads and exogenous IL-2(DYNABEADS), or with various APC-MS formulations. FIG. 30F depicts theFACS quantification of CD4and CD8 single positive cells among livesingle CD3+ cells, in samples expanded for 14 days either with DYNABEADSor with various APC-MS formulations. FIG. 30G depicts the FACSquantification of cells co-expressing PD-1 and LAG-3 among live singlecells, in samples expanded either with DYNABEADS or with various APC-MSformulations. Data in FIGS. 30F and 30G represent mean±s.d. of threeexperimental replicates and are representative of at least twoindependent experiments. Data in FIG. 30E represent mean±s.d. of atleast three different donor samples from two independent experiments.Data in FIGS. 30F and 30G represent mean±s.d. of three different donorsamples and are representative of at least two independent experiments.**p<0.01, ***p<0.001.

FIG. 31 depicts representative FACS plots of CD4 and CD8 expression onpolyclonally expanded primary mouse T cells. Representative FACS plotsshowing CD4 and CD8 expression on live single cells that werepolyclonally expanded with either APC-MS or DYNABEADS. Flow data weregated on Fluorescence Minus One (FMO) controls for each sample, at eachtimepoint. Data is representative of at least two independentexperiments.

FIGS. 32A, 32B, 32C and 32D depict the extended phenotypiccharacterization of polyclonally expanded primary mouse T cells. FIG.32A depicts the FACS quantification of Granzyme B positive cells amonglive single CD8+ cells, in samples expanded either with DYNABEADS orwith APC-MS (left), and representative FACS plots (right). FIG. 32Bdepicts the FACS quantification of FoxP3 positive cells among livesingle CD4+ cells, in samples expanded either with DYNABEADS or withAPC-MS. FIGS. 32C and 32D shows representative FACS plots showing PD-1expression on live single cells, as a function of CD8 expression. Flowdata were gated on Fluorescence Minus One (FMO) controls for eachsample, at each timepoint. Data represent mean±s.d. of threeexperimental replicates and are representative of at least twoindependent experiments.

FIG. 33 shows adhesion molecule expression on polyclonally expandedprimary human T cells. FACS quantification of live single cellsco-expressing CD62L and CCR7, in samples expanded either with DYNABEADSor with various APC-MS formulations. (F1) APC-MS presenting αCD3andαCD28 saturating 1 mol % biotinylated lipid, input at 333 μg/ml of MSRsto initial culture, (F2) APC-MS presenting αCD3and αCD28 saturating 1mol % biotinylated lipid, input at 33 μg/ml of MSRs to initial culture,(F3) APC-MS presenting αCD3and αCD28 saturating 0.1 mol % biotinylatedlipid, input at 333 μg/ml of MSRs to initial culture, and (F4) APC-MSpresenting αCD3and αCD28 saturating 0.1 mol % biotinylated lipid, inputat 33 μg/ml of MSRs to initial culture. Data represents mean±s.d. ofthree different donor samples and is representative of at least twoindependent experiments.

FIGS. 34A, 34B, 34C, 34D, and 34E depict antigen-specific expansion ofprimary mouse T cells. FIG. 34A shows representative brightfieldmicroscopy images of primary CD8+OT-J T cells cultured for two days withAPC-MS presenting an irrelevant peptide (SVYDFFVWL (SEQ ID NO: 3); left)or the relevant peptide (SIINFEKL (SEQ ID NO: 4); right) in H-2K(b).Scale bar=100 μm. FIG. 34B shows the expansion of primary CD8+OT-I Tcells that were either untreated (mock), or cultured with various APC-MSformulations. (F1) APC-MS presenting SIINFEKL (SEQ ID NO: 4)/H-2K(b) andαCD28 saturating 1 mol % biotinylated lipid, input at 333 μg/ml of MSRsto initial culture, (F2) APC-MS presenting SIINFEKL (SEQ ID NO:4)/H-2K(b) and αCD28 saturating 1 mol % biotinylated lipid, input at 33μg/ml of MSRs to initial culture, (F3) APC-MS presenting SIINFEKL (SEQID NO: 4)/H-2K(b) and αCD28 saturating 0.1 mol % biotinylated lipid,input at 333 μg/ml of MSRs to initial culture, and (F4) APC-MSpresenting SIINFEKL (SEQ ID NO: 4)/H-2K(b) and αCD28 saturating 0.1 mol% biotinylated lipid, input at 33 μg/ml of MSRs to initial culture. FIG.34C depicts the FACS quantification of IFNγ and TNFα expression by livesingle CD8+ OT-I T cells expanded for 13 days with various APC-MSformulations and then co-cultured with B16-F10 cells that were eithermock pulsed (−), or pulsed with SIINFEKL (SEQ ID NO: 4) peptide (+).FIG. 34D depicts the quantification of in vitro killing of mock-pulsed(−) or SIINFEKL (SEQ ID NO: 4)-pulsed (+) B16-F10 target cells by CD8+OT-I T cells that were expanded for 13 days with various APC-MSformulations, and then co-cultured at various effector:target cellratios. FIG. 34E depicts the quantification of IFNγ secretion by CD8+OT-I T cells expanded for 13 days with various APC-MS formulations inresponse to co-culture at various effector:target cell ratios withB16-F10 cells that were either mock pulsed (pep−), or pulsed withSIINFEKL (SEQ ID NO: 4) peptide (pep+). Data in FIGS. 34B, 34C, 34D, and34E represent mean±s.d. of three experimental replicates and arerepresentative of at least two independent experiments.

FIGS. 35A, 35B, 35C, 35D and 35E show the extended characterization ofprimary human T cells expanded with antigen-specific APC-MSformulations. FIG. 35A shows the total expansion of primary human CD8+ Tcell isolates that were mock treated (30 U/ml IL-2), or cultured withAPC-MS (loaded with pMHC, αCD28, IL-2) either presenting the CLG or GLCpeptide in HLA-A2. Data for mock-treated cells only available for days 0and 7. FIGS. 35B, 35C and 35D show the quantification of IFNγ secretionof CD8+ T cell isolates that were mock treated (30 U/ml IL-2), orcultured with APC-MS presenting either the CLG peptide (APC-MS CLG) orGLC peptide (APC-MSGLC), following co-culture with T2 cells that wereeither unpulsed (peptide−) (FIG. 35B), pulsed with CLG peptide (+CLGpeptide) (FIG. 35C), or pulsed with GLC peptide (+GLC peptide) (FIG.35D). Data for mock-treated cells only available for day 7. FIG. 35Eshows representative FACS plots showing IFNγ and TNFα expression, ofCD8+ T cell isolates that were cultured with APC-MS presenting eitherthe CLG peptide (APC-MS/CLG) or GLC peptide (APC-MS/GLC), followingco-culture with T2 cells that were either unpulsed (no peptide; top),pulsed with CLG peptide (+CLG peptide; middle), or pulsed with GLCpeptide (+GLC peptide; bottom). Data in FIGS. 35A and 35B representmean±s.d. of three experimental replicates and are representative of twoexperiments with two different donor samples.

FIGS. 36A, 35B, 36C, 36D, 36E, 36F, 36G, 36H, 36I, 36J, 36K, 36L, 36M,and 36N show the antigen-specific expansion of primary human T cells.FIGS. 36A, 36B, 36C, 36D, 36E, 36F, 36G, 36H, 36I, and 36J depict theantigen-specific expansion of primary human T cells from CD8+ T cellisolates. FIGS. 36A, 36B and 36D depict the tetramer analysis of liveCD8+ single cells specific for the EBV-derived peptides CLGGLLTMV (SEQID NO: 1) (CLG; FIGS. 36A and 36B) and GLCTLVAML (SEQ ID NO. 2) (GLC;FIGS. 36D and 36E). Representative FACS plots with numbers in gatesdenoting the percent of live single CD8+ cells that are positive for therespective tetramer (FIGS. 36A and 36D), and quantification of FACS dataat various timepoints (FIGS. 36B and 36E), of primary HLA-A2+ human CD8+T cells that were mock treated (30 U/ml IL-2), or cultured with APC-MS(loaded with pMHC, αCD28, IL-2) either presenting the CLG or GLC peptidein HLA-A2. Data for mock-treated cells only available for days 0 and 7.FIG. 36F shows the expansion of primary human CD8+ T cells specific forCLG (FIG. 36C) or GLC (FIG. 36F) that were either mock treated, orcultured with APC-MS either presenting the CLG or GLC peptide in HLA-A2.Data for mock-treated cells only available for days 0 and 7. FIGS. 36G,36H and 36I depict the frequencies of TNFα+IFNγ+ cells among live singleCD8+ T cells that were mock treated, or cultured with APC-MS eitherpresenting the CLG or GLC peptide in HLA-A2, following co-culture withT2 cells that were either unpulsed (peptide-; FIG. 36G), pulsed with CLGpeptide (+CLG peptide; FIG. 36H), or pulsed with GLC peptide (+GLCpeptide; FIG. 36I). Data for mock-treated cells only available for day7. FIG. 36J shows the quantification of in vitro killing of T2 targetcells that were mock-pulsed (no peptide), or pulsed with either the CLGpeptide (+CLG) or GLC peptide (+GLC), by primary human CD8+ T cellsexpanded for 14 days with APC-MS either presenting the CLG or GLCpeptide in HLA-A2. FIGS. 36K, 36L, 36M and 36N show the antigen-specificexpansion of primary human T cells from PBMCs. FIG. 36K depicts thefrequency of GLC-specific cells among live single CD8+ T cells, withinPBMCs cultured for 7 days in 30 U/ml IL-2 (mock), or with APC-MSpresenting the GLC peptide in HLA-A2. FIG. 36L shows the number ofGLC-specific CD8+ T cells within PBMCs cultured for 7 days in 30 U/mlIL-2 (mock), or with APC-MS presenting the GLC peptide in HLA-A2.Numbers above bars denote fold expansion (mean±s.d.). FIGS. 36M and 36Nshow the frequency of TNFα+IFNγ+ cells among live single CD8+ T cells(FIG. 36M), and IFNγ secretion (FIG. 36N), from PBMCs that were culturedfor 7 days in 30 U/ml IL-2 (mock), or with APC-MS presenting the GLCpeptide in HLA-A2, following co-culture with T2 cells that were eitherunpulsed (no peptide), pulsed with CLG peptide (+CLG), or pulsed withGLC peptide (+GLC). All data represent mean±s.d. of three experimentalreplicates and are representative of two experiments with two differentdonor samples.

FIG. 37 depicts the degradation of APC-MS scaffold in vitro. APC-MS (167μg) presenting αCD3/αCD28 (1% biotinylated lipid) and releasing IL-2 wascultured with primary mouse T cells (25×10⁴ T cells/167 μg APC-MS). Atvarious timepoints, cultures were centrifuged at 700 rcf for 5 min, andSi content in pellets was quantified via inductively coupled plasmaoptical emission spectrometry (ICP-OES; Galbraith Laboratories). Si isundetectable in culture pellets by 1 week after starting culture.

FIG. 38 shows the controlled release of diverse soluble immune-directingpayloads from APC-MS. 4 APC-MS were generated, each comprising 2 μg ofeither IL-2, IL-21, TGFβ or IL-15SA loaded into 500 μg APC-MS prior tolipid coating. Samples were thoroughly washed to remove unloaded proteinand subsequently maintained at 37° C. for up to 28 days. Payload releaseover time was evaluated via ELISA.

FIGS. 39A and 39B depict fluorescence recovery after photobleaching(FRAP) experiments using MSR-SLBs containing 10%carboxyfluoresceinheadgroup-tagged lipid. FIG. 39A are representativeimages of three independent FRAP events. Images showfluorescently-tagged MSR-SLB before photobleaching (left), immediatelyafter photobleaching (middle), and after fluorescence recovery (right).Photobleached regions are indicated by red arrows. FIG. 39B shows thequantification of fluorescence recovery over time. Fluorescence recoveryof 8 independent photobleaches on different MSR-SLBs are shown in dashedblack and the average trend is shown in solid.

FIGS. 40A, 40B, 40C, and 40D depict the results of T-cell expansionexperiments performed using APC-MSs as compared to DYNABEADs, whereinthe amount of DYNABEADs was normalized to comprise the same amount ofanti-CD3 and anti-CD28 antibodies as the APC-MSs. FIG. 40A.Bicinchoninic acid assay (BCA) analysis for total protein quantificationperformed to determine the amount of protein bound on the surface ofcommercial mouse or human CD3/CD28 T cell activator DYNABEADS. DYNABEADstock solutions were washed thoroughly, and DYNABEAD antibody load wasevaluated via BCA assay. DYNABEADs targeted to mouse and human T-cellswere found to have similar antibody loads (˜20 μg/ml). On a per cellbasis, a DYNABEAD:cell of 5:1 ratio (condition D-B) corresponded to thesame dose of anti-CD28/anti-CD3 antibodies as APC-MS presenting 0.1% Tcell cues input at 16.7 μg (condition M-D). FIG. 40B. Dose-dependentexpansion of primary mouse T-cells was observed with APC-MS over 13-dayculture period, but not with DYNABEADs within the dose range tested.APC-MS significantly promoted enhanced T cell expansion compared toDYNABEADS presenting the same amount of anti-CD3 and anti-CD28antibodies (see condition M-D vs D-B). FIG. 40C. Despite greaterexpansion, cells expanded with APC-MS condition M-D did not showenhanced co-expression of exhaustion markets PD-1 and LAG-3 as comparedto cells expanded with DYNABEADs presenting the same amount of anti-CD3and anti-CD28 antibodies (condition D-B). FIG. 40D. T cells expandedwith low-to-moderate doses of DYNABEADs showed primarily CD4-biasedskewing (conditions D-A, D-B). When DYNABEADs were added at extremelyhigh doses, moderate CD8-biased skewing was observed (condition D-C). Incontrast, APC-MS tended to show heavy CD8-biased skewing with the degreeof skewing dependent on the formulation of the APC-MS. Data in FIGS.40B, 40C and 40D represent mean±s.d. of samples from four different miceand are representative of at least two independent experiments.***p<0.001, (b) analyzed using two-way ANOVA, followed by Tukey HSDpost-hoc test.

FIGS. 41A and 41B depict the results of experiments performed toevaluate the effect on primary mouse T-cell expansion of IL-2 dose andsustained release from APC-MS as compared to DYNABEADs. FIG. 41A showsthe expansion of primary mouse T cells treated with either APC-MS loadedwith IL-2 (M-D), APC-MS and IL-2 added to media (M-D bIL2); DYNABEADs(D-B) or DYNABEADs and IL-2 added to media (D-B bIL-2). D-B: DYNABEAD5:1; D-B-bIL-2: DYNABEAD 5:1+IL-2 bolus; M-D: 0.1% T cell cues/1:10×material/loaded IL-2; M-S/bIL-2: 0.1% T cell cues/1L10× material/IL-2bolus. FIG. 41B shows the co-expression of exhaustion markers PD-1 andLAG-3 in primary mouse T-cells cells expanded with either APC-MS loadedwith IL-2 (M-D); APC-MS and IL-2 added to media (M-D bIL2); DYNABEADs(D-B); or DYNABEADs and IL-2 added to media (D-B bIL-2). Data representmean±s.d. of samples from four different mice and are representative ofat least two independent experiments. ***p<0.001, analyzed using two-wayANOVA, followed by Tukey HSD post-hoc test.

FIGS. 42A and 42B depict the attachment of azide-labeled IgG toDBCO-presenting MSR-SLBs via click-chemistry conjugation. FIG. 42A.Varying amounts of azide-modified IgG (as indicated) were incubated withMSR-SLBs containing varying amounts of DBCO-modified lipid (asindicated). Values above bars represent ug of azide-modified IgG thatwas attached to MSR-SLBs. FIG. 42B shows the broader dose titration ofazide-modified IgG input to MSR-SLBs containing varying amounts ofDBCO-modified lipid. nIgG represents IgG that was not azide-modified.Values above bars represent g of azide-modified IgG that was attached tothe MSR-SLBs.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides a solution to the problem of manipulatingT-cells. Specifically, the present invention provides antigen presentingcell-mimetic scaffolds (APC-MS), which are useful in the manipulation ofsuch cells. The scaffolds include mesoporous silica rods (MSR), whichincorporate or are coated with a continuous, fluid supported lipidbilayer (SLB) thereby forming MSR-SLB scaffolds. The MSR-SLB scaffoldfurther contains a plurality of T-cell activating and T-cellco-stimulatory molecules, along with a plurality of T-cell homeostaticagents, which together make up a structure that mimicsantigen-presenting cells (APC) and allows the scaffolds to elicitvarious effector functions on target cells, e.g., T-cells. In someembodiments, the scaffold mediates these effects via direct or indirectinteraction between the cell surface molecules residing in target cellsand the various binding partners presented by the scaffolds. Dependingon the application for which the scaffold is used, the scaffoldregulates survival and growth of the targeted cells through the physicalor chemical characteristics of the scaffold itself. Depending uponapplication, the scaffold composition may be modified to contain certainactivating and co-stimulatory signals, as well as homeostatic signalingmolecules, which act together to mediate various effector functions,e.g., activation, division, promote differentiation, growth, expansion,reprogramming, anergy, quiescence, senescence, apoptosis or death, oftarget cells. In these applications, the scaffolds were found tosurprisingly improve cell metabolic activity and growth of targetedcells. Moreover, the improvement in growth and metabolic activityconferred by the scaffolds of the invention was unexpectedly superior toexisting platforms, such as magnetic beads.

In order to permit manipulation of specific cells, such as T-cells, thepermeability of the scaffold composition may be regulated, for example,by selecting or engineering a material for greater or smaller pore size,density, polymer cross-linking, stiffness, toughness, ductility, orelasticity. The scaffold composition may contain physical channels orpaths through which targeted cells interact with the scaffold and/ormove into a specific compartment or region of the scaffold. Tofacilitate the compartmentalization, the scaffold composition may beoptionally organized into compartments or layers, each with a differentpermeability, so that cells are sorted or filtered to allow access toonly a certain sub-population of cells. Sequestration of target cellpopulations in the scaffold may also be regulated by the degradation,de- or re-hydration, oxygenation, chemical or pH alteration, or ongoingself-assembly of the scaffold composition. Following their capture, thetargeted cells may be allowed to grow or expand within the scaffold withthe help of stimulatory molecules, cytokines, and other co-factorspresent in the scaffold. In other instances, non-targeted cells whichhave otherwise infiltrated the scaffold may be rejected or removed usingnegative selection agents.

The cells that are contained or sequestered within the scaffolds of theinvention are primarily immune cells. In certain embodiments, theinvention relates to scaffolds for sequestering and/or manipulating Tcells. In other embodiments, the invention relates to scaffolds that arepermeable to other lymphocytes, e.g., B-cells. Yet in other embodiments,the invention relates to a combination of scaffolds, e.g., a combinationof T-cell scaffolds and B-cell scaffolds. The immune cells, e.g.,T-cells, are optionally harvested and analyzed to identify distinctsub-populations that are useful in the diagnosis or therapy of diseases.The harvested cells may also be reprogrammed or expanded for developingcompositions or formulations that are to be used in therapy.

The invention is further described in more detail in the subsectionsbelow.

I. Antigen Presenting Cell-Mimetic Scaffolds (APC-MS)

In one embodiment, the present invention provides antigen-presentingcell-mimetic scaffolds (APC-MS). The scaffolds contain a base layercomprising high surface area mesoporous silica micro-rods (MSR); acontinuous, fluid supported lipid bilayer (SLB) layered on the MSR baselayer; a plurality of T-cell activating molecules and T-cellco-stimulatory molecules adsorbed onto the scaffold; and a plurality ofT-cell homeostatic agents adsorbed onto the scaffold.

A. Mesoporous Silica

In one embodiment, the components of the scaffolds of the inventioninclude mesoporous silica. Mesoporous silica is a porous body withhexagonal close-packed, cylinder-shaped, uniform pores. This material issynthesized by using a rod-like micelle of a surfactant as a template,which is formed in water by dissolving and hydrolyzing a silica sourcesuch as alkoxysilane, sodium silicate solution, kanemite, silica fineparticle in water or alcohol in the presence of acid or basic catalyst.See, US Pub. No. 2015-0072009 and Hoffmann et al., Angewandte ChemieInternational Edition, 45, 3216-3251, 2006. Many kinds of surfactantssuch as cationic, anionic, and nonionic surfactants have been examinedas the surfactant and it has been known that generally, an alkyltrimethylammonium salt of cationic surfactant leads to a mesoporoussilica having the greatest specific surface area and a pore volume. See,U.S. Publication No. 2013/0052117 and Katiyar et al. (Journal ofChromatography 1122 (1-2): 13-20). The terms “mesoscale,” “mesopore,”“mesoporous” and the like, as used in this specification, may refer tostructures having feature sizes in the range of 5 nm to 100 nm, inparticular in the range of 2 nm to 50 nm. Hence, in some embodiments, amesoporous material includes pores, which may be ordered or randomlydistributed, having a diameter in the range of 5 nm to 100 nm.

The mesoporous silica used in the scaffolds of the invention may beprovided in various forms, e.g., microspheres, irregular particles,rectangular rods, round nanorods, etc., although structured rod forms(MSR) are particularly preferred. The particles can have variouspre-determined shapes, including, e.g., a spheroid shape, an ellipsoidshape, a rod-like shape, or a curved cylindrical shape. Methods ofassembling mesoporous silica to generate microrods are known in the art.See, Wang et al., Journal of Nanoparticle Research, 15:1501, 2013. Inone embodiment, mesoporous silica nanoparticles are synthesized byreacting tetraethyl orthosilicate with a template made of micellar rods.The result is a collection of nano-sized spheres or rods that are filledwith a regular arrangement of pores. The template can then be removed bywashing with a solvent adjusted to the proper pH. In this example, afterremoval of surfactant templates, hydrophilic silica nanoparticlescharacterized by a uniform, ordered, and connected mesoporosity areprepared with a specific surface area of, for example, about 600 m²/g toabout 1200 m²/g, particularly about 800 m²/g to about 1000 m²/g andespecially about 850 m²/g to about 950 m²/g.

In another embodiment, the mesoporous particle could be synthesizedusing a simple sol-gel method or a spray drying method. Tetraethylorthosilicate is also used with an additional polymer monomer (as atemplate). In yet another embodiment, one or more tetraalkoxy-silanesand one or more (3-cyanopropyl)trialkoxy-silanes may be co-condensed toprovide the mesoporous silicate particles as rods. See, US PublicationNos. 2013-0145488, 2012-0264599 and 2012-0256336, which are incorporatedby reference.

The mesoporous silica rods may comprise pores of between 2-50 nm indiameter, e.g., pores of between 2-5 nm, 10-20 nm, 10-30 nm, 10-40 nm,20-30 nm, 30-50 nm, 30-40 nm, 40-50 nm. In particular embodiments, themicrorods comprise pores of approximately 5 nm, 6 nm, 7 nm, 8 nm, 9 nm,10 nm, 11 nm, 12 nm, or more in diameter. The pore size may be altereddepending on the type of application.

In another embodiment, the length of the micro rods is in the micrometerrange, ranging from about 5 μm to about 500 μm. In one example, themicrorods comprise a length of 5-50 μm, e.g., 10-20 μm, 10-30 μm, 10-40μm, 20-30 μm, 30-50 μm, 30-40 μm, 40-50 μm. In other embodiment, therods comprise length of 50 μm to 250 μm, e.g., about 60 μm, 70 μm, 80μm, 90 m, 100 μm, 120 μm, 150 μm, 180 μm, 200 μm, 225 μm, or more. Forrecruitment of cells, it may be preferable to employ MSR compositionshaving a higher aspect ratio, e.g., with rods comprising a length of 50μm to 200 μm, particularly a length of 80 μm to 120 μm, especially alength of about 100 μm or more.

In yet another embodiment, the MSR provide a high surface area forattachment and/or binding to target cells, e.g., T-cells. Methods ofobtaining high surface area mesoporous silicates are known in the art.See, e.g., U.S. Pat. No. 8,883,308 and US Publication No. 2011-0253643,the entire contents of which are incorporated by reference herein. Inone embodiment, the high surface area is due to the fibrous morphologyof the nanoparticles, which makes it possible to obtain a highconcentration of highly dispersed and easily accessible moieties on thesurface. In certain embodiments, the high surface area MSRs have asurface area of at least about 100 m²/g, at least 150 m²/g, or at least300 m²/g. In other embodiments, the high surface area MSRs have asurface area from about 100 m²/g to about 1000 m²/g, including allvalues or sub-ranges in between, e.g., 50 m²/g, 100 m²/g, 200 m²/g, 300m²/g, 400 m²/g, 600 m²/g, 800 m²/g, 100-500 m²/g, 100-300 m²/g, 500-800m²/g or 500-1000 m²/g.

B. Lipids

The scaffolds of the invention comprise a continuous, fluid supportedlipid bilayer (SLB) on the MSR base layer. The term “lipid” generallydenotes a heterogeneous group of substances associated with livingsystems which have the common property of being insoluble in water, canbe extracted from cells by organic solvents of low polarity such aschloroform and ether. In one embodiment, “lipid” refers to any substancethat comprises long, fatty-acid chains, preferably containing 10-30carbon units, particularly containing 14-23 carbon units, especiallycontaining 16-18 carbon units.

In one embodiment, the lipid is provided as a monolayer. In anotherembodiment, the lipid is provided as a bilayer. A lipid bilayer is athin polar membrane made of two layers of lipid molecules. Preferably,the lipid bilayer is fluid, wherein individual lipid molecules able todiffuse rapidly within the monolayer. The membrane lipid molecules arepreferably amphipathic.

In one embodiment, the lipid layers are continuous bilayers, e.g.,resembling those found in natural biological membranes such as cellularplasma membranes. In another embodiment, the lipid is provided in theform of a supported bilayer (SLB). An SLB is a planar structure sittingon a solid support, e.g., mesoporous silica rods (MSR). In such anarrangement, the upper face of the supported bilayer is exposed, whilethe inner face of the supported bilayer is in contact with the support.MSR-SLB scaffolds are stable and remain largely intact even when subjectto high flow rates or vibration and can withstand holes, e.g., holesthat are aligned with the pores of the mesoporous silica base layer.Because of this stability, experiments lasting weeks and even months arepossible with supported bilayers. SLBs are also amenable tomodification, derivatization and chemical conjugation with many chemicaland/or biological moieties.

In one embodiment, the SLB may be immobilized on the MSR base layerusing any known methods, including covalent and non-covalentinteractions. Types of non-covalent interactions include, for example,electrostatic interactions, van der Waals' interactions, π-effects,hydrophobic interactions, etc. In one embodiment, the lipids areadsorbed on the MSR base layer. In another embodiment, the SLBs areattached or tethered to the MSR base layer via covalent interactions.Methods for attaching lipids to silicates are known in the art, e.g.,surface absorption, physical immobilization, e.g., using a phase changeto entrap the substance in the scaffold material. In one embodiment, thelipid bilayers are layered onto the MSR base layer. For example, a lipidfilm (containing for example, a solution of DPPC/cholesterol/DSPE-PEG ata molar ratio of 77.5:20:2.5 in chloroform) may be spotted onto themesoporous silica and the solvent is evaporated using a rotaryevaporator. See Meng et al., ACS Nano, 9 (4), 3540-3557, 2015. In oneembodiment, the lipid bilayer can be prepared, for example, by extrusionof hydrated lipid films through a filter with pore size of, for example,about 100 nm, using standard protocols. The filtered lipid bilayer filmscan then be fused with the porous particle cores, for example, by apipette mixing.

Alternatively, covalent coupling via alkylating or acylating agents maybe used to provide a stable, structured and long-term retention of theSLB on the MSR layer. In such embodiments, the lipid bilayers may bereversibly or irreversibly immobilized onto the MSR layers using knowntechniques. For example, the MSR base layer can be hydrophilic and canbe further treated to provide a more hydrophilic surface, e.g., withammonium hydroxide and hydrogen peroxide. The lipid bilayer can befused, e.g., using known coupling techniques, onto the porous MSR baselayer to form the MSR-SLB scaffolds. The scaffolds may be furtherprocessed and derivatized with additional moieties to allow attachmentand/or immobilization of other secondary agents onto the structure.

Accordingly, in one embodiment, the instant invention provides MSR-SLBscaffolds, wherein the SLB component is a phospholipid. Representativeexamples of such lipids include, but are not limited to, amphotericliposomes described in U.S. Pat. Nos. 9,066,867 and 8,3676,28. Forexample, the lipid bilayer may comprise a lipid selected fromdimyristoylphosphatidylcholine (DMPC), dipalmitoylphosphatidylcholine(DPPC), distearoylphosphatidylcholine (DSPC),palmitoyl-oleoylphosphatidylcholine (POPC), dioleoylphosphatidylcholine(DOPC), dioleoyl-phosphatidylethanolamine (DOPE),dimyristoyl-phosphatidylethanolamine (DMPE) anddipalmitoyl-phosphatidylethanolamine (DPPE) or a combination thereof. Insome embodiments, the lipid bilayer comprises a lipid composition thatmimics the lipid composition of a mammalian cell membrane (e.g., a humancell plasma membrane). The lipid composition of many mammalian cellmembranes have been characterized and are readily ascertainable by oneof skill in the art (see, e.g., Essaid et al. Biochim. Biophys. Acta1858(11): 2725-36 (2016), the entire contents of which are incorporatedherein by reference). The composition of the lipid bilayer may bealtered to modify the charge or fluidity of the lipid bilayer. In someembodiments, the lipid bilayer comprises cholesterol. In someembodiments, the lipid bilayer comprises a sphingolipid. In someembodiments, the lipid bilayer comprises a phospholipid. In someembodiments, the lipid is a phosphatidylethanolamine, aphosphatidylcholine, a phosphatidylserine, a phosphoinositide aphosphosphingolipid with saturated or unsaturated tails comprising 6-20carbons, or a combination thereof.

In another embodiment, the lipid is DIYNE PC lipid. Representativeexamples of such lipids include, but are not limited to,1-Palmitoyl-2-10,12 Tricosadiynoyl-sn-Glycero-3-Phosphocholine(16:0-23:2 DIYNE PC) and1,2-bis(10,12-tricosadiynoyl)-SN-Glycero-3-Phosphocholine (23:2 DiynePC).

In one embodiment, the MSR-SLB scaffold of the invention retains acontinuous, fluid architecture for at least 1 day, at least 2 days, atleast 3 days, at least 4 days, at least 5 days, at least 6 days, atleast 7 days, at least 8 days, at least 9 days, at least 10, at least 11days, at least 12 days, at least 13 days, at least 14 days, at least 15days, at least 16 days, at least 17 days, at least 18 days, at least 19days, at least 20 days, at least 21 days, at least 25 days, at least 30days, at least 35 days, at least 40 days, at least 50 days, or more.

The architecture of the MSR-SLB scaffold may be studied with any knowntechniques, including, the microscopic visualization techniquesillustrated in the Examples below.

C. Functional Molecules

In an embodiment of the instant invention, the MSR-SLB scaffold maycontain one or more functional molecules. The term “functional molecule”includes any molecule which possesses biologically desirable properties.In the context of the invention, examples of such functional moleculesinclude proteins, peptides, antigens, antibodies, DNA, RNA,carbohydrates, haptens, and other small molecules, e.g., drugs. In oneembodiment, the functional molecule is a T-cell activating molecule. Inanother embodiment, the functional molecule is a T-cell co-stimulatorymolecule. Still further, in one embodiment, the functional molecule is aT-cell homeostatic agent. In certain embodiments, the MSR-SLB scaffoldscomprise a plurality of functional molecules, e.g., at least one T-cellactivating molecule, at least one T-cell co-stimulatory molecule, and atleast one T-cell homeostatic agent.

T-Cell Activating Molecules

In one embodiment, the instant invention provides for MSR-SLB scaffoldscontaining a plurality of T-cell activating molecules. These activatingmolecules may mediate direct, indirect, or semi-direct activation of atarget population of T-cells. See, Benichou et al., Immunotherapy, 3(6):757-770, 2011. Preferably, the T-cell activating molecules mediatedirect activation of T-cells.

In one embodiment, the instant invention provides for MSR-SLB scaffoldscontaining molecules which directly activate T-cells, e.g., via bindingto cell surface receptors on target T-cells. Particularly, the directactivation may be mediated via cluster of differentiation-3 (CD3), whichis a T-cell co-receptor that helps to activate cytotoxic T-cells. Inanother embodiment, T-cells may be directly activated withoutconcomitant participation of CD3, e.g., in a CD3-independent manner.

In one embodiment, the target T-cells are activated in a CD3-dependentmanner. It is generally believed that T cell activation requires a Tcell receptor (TCR) to recognize its cognate peptide in the context ofan MHC molecule. In addition, the association of CD3 with theTCR-peptide-MHC complex transmits the activation signal to intracellularsignaling molecules to initiate a signaling cascade in the T cell. See,Ryan et al., Nature Reviews Immunology 10, 7, 2010. The CD3 receptorcomplex found on T-cells contains a CD3γ chain, a CD3δ chain, and twoCD3ε chains, which associate with TCR and the ζ-chain (zeta-chain;CD247) to generate an activation signal in T cells. The TCR, ζ-chain,and CD3 molecules together constitute the T cell receptor (TCR) complex.Binding of an activating molecule, e.g., an antibody, to one or more ofthe members of the TCR complex may activate the T-cell.

In one embodiment, the T-cell activating molecule is an antibody or anantigen binding fragment thereof. Where the T-cell activating moleculeacts in a CD3-dependent manner, the T-cell activating molecule ispreferably an anti-CD3 antibody or an antigen-binding fragment thereof.In another embodiments, the T-cell activating molecule may include, forexample, an anti-CD2 antibody or an antigen-binding fragment thereof, ananti-CD47 antibody or an antigen-binding fragment thereof,anti-macrophage scavenger receptor (MSR1) antibody or an antigen-bindingfragment thereof, an anti-T-cell receptor (TCR) antibody or anantigen-binding fragment thereof, etc. In another embodiment, the T-cellactivating molecule is a major histocompatibility complex (MHC) moleculeor a multimer thereof that is optionally loaded with an MHC peptide.Still further, the T-cell activating molecule is a conjugate containingMHC and immunoglobulin (Ig) or a multimer thereof.

The term “antibody”, as used herein, broadly refers to anyimmunoglobulin (Ig) molecule comprised of four polypeptide chains, twoheavy (H) chains and two light (L) chains, or any functional fragment,mutant, variant, or derivation thereof, which retains the essentialepitope binding features of an Ig molecule. Such mutant, variant, orderivative antibody formats are known in the art. Non-limitingembodiments of which are discussed herein. In one embodiment, the T-cellactivating antibody used in the compositions and methods of thedisclosure is the anti-CD3 antibody selected from the group consistingof muromonab (OKT3), otelixizumab (TRX4), teplizumab (hOKT3γ1(Ala-Ala)),visilizumab, an antibody recognizing 17-19 kDa ε-chain of CD3 within theCD3 antigen/T cell antigen receptor (TCR) complex (HIT3a), and anantibody recognizing a 20 kDa subunit of the TCR complex within CD3e(UCHT1), or an antigen-binding fragment thereof. Other anti-CD3antibodies, including, antigen-binding fragments thereof are describedin US patent pub. No. 2014-0088295, which is incorporated by reference.

Embodiments of the invention include “full-length” antibodies. In afull-length antibody, each heavy chain is comprised of a heavy chainvariable region (abbreviated herein as HCVR or VH) and a heavy chainconstant region. The heavy chain constant region is comprised of threedomains, CH1, CH2 and CH3. Each light chain is comprised of a lightchain variable region (abbreviated herein as LCVR or VL) and a lightchain constant region. The light chain constant region is comprised ofone domain, CL. The VH and VL regions can be further subdivided intoregions of hypervariability, termed complementarity determining regions(CDR), interspersed with regions that are more conserved, termedframework regions (FR). Each VH and VL is composed of three CDRs andfour FRs, arranged from amino-terminus to carboxy-terminus in thefollowing order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. Immunoglobulinmolecules can be of any type (e.g., IgG, IgE, IgM, IgD, IgA and IgY),class (e.g., IgG 1, IgG2, IgG 3, IgG4, IgA1 and IgA2) or subclass.

The term “antigen-binding portion” of an antibody (or simply “antibodyportion”), as used herein, refers to one or more fragments of anantibody that retain the ability to specifically bind to an antigen(e.g., IL-13). It has been shown that the antigen-binding function of anantibody can be performed by fragments of a full-length antibody. Suchantibody embodiments may also be bispecific, dual specific, ormulti-specific formats; specifically binding to two or more differentantigens. Examples of binding fragments encompassed within the term“antigen-binding portion” of an antibody include (i) a Fab fragment, amonovalent fragment consisting of the VL, VH, CL and CH1 domains; (ii) aF(ab′)₂ fragment, a bivalent fragment comprising two Fab fragmentslinked by a disulfide bridge at the hinge region; (iii) a Fd fragmentconsisting of the VH and CH1 domains; (iv) a Fv fragment consisting ofthe VL and VH domains of a single arm of an antibody, (v) a dAb fragment(Ward et al., (1989) Nature 341:544-546, Winter et al., PCT publicationWO 90/05144 A1 herein incorporated by reference), which comprises asingle variable domain; and (vi) an isolated complementarity determiningregion (CDR). Furthermore, although the two domains of the Fv fragment,VL and VH, are coded for by separate genes, they can be joined, usingrecombinant methods, by a synthetic linker that enables them to be madeas a single protein chain in which the VL and VH regions pair to formmonovalent molecules (known as single chain Fv (scFv); see e.g., Bird etal. (1988) Science 242:423-426; and Huston et al. (1988) Proc. Natl.Acad. Sci. USA 85:5879-5883). Such single chain antibodies are alsointended to be encompassed within the term “antigen-binding portion” ofan antibody. Other forms of single chain antibodies, such as diabodiesare also encompassed. Diabodies are bivalent, bispecific antibodies inwhich VH and VL domains are expressed on a single polypeptide chain, butusing a linker that is too short to allow for pairing between the twodomains on the same chain, thereby forcing the domains to pair withcomplementary domains of another chain and creating two antigen bindingsites (see e.g., Holliger et al., Proc. Natl. Acad. Sci. USA90:6444-6448 (1993); Poljak et al., Structure 2:1121-1123 (1994)). Suchantibody binding portions are known in the art (Kontermann and Dubeleds., Antibody Engineering (2001) Springer-Verlag. New York. 790 pp.(ISBN 3-540-41354-5).

“Antibody fragments” comprise only a portion of an intact antibody,wherein the portion preferably retains at least one, and typically mostor all, of the functions normally associated with that portion whenpresent in an intact antibody. In one embodiment, an antibody fragmentcomprises an antigen binding site of the intact antibody and thusretains the ability to bind antigen. In another embodiment, an antibodyfragment, for example one that comprises the Fc region, retains at leastone of the biological functions normally associated with the Fc regionwhen present in an intact antibody, such as FcRn binding, antibodyhalf-life modulation, ADCC function and complement binding. In oneembodiment, an antibody fragment is a monovalent antibody that has an invivo half-life substantially similar to an intact antibody. For example,such an antibody fragment may comprise on antigen binding arm linked toan Fc sequence capable of conferring in vivo stability to the fragment.

The term “antibody construct” as used herein refers to a polypeptidecomprising one or more the antigen binding portions of the disclosurelinked to a linker polypeptide or an immunoglobulin constant domain.Linker polypeptides comprise two or more amino acid residues joined bypeptide bonds and are used to link one or more antigen binding portions.Such linker polypeptides are well known in the art (see e.g., Holligeret al., Proc. Natl. Acad. Sci. USA 90:6444-6448 (1993); Poljak et al.,Structure 2:1121-1123 (1994)). An immunoglobulin constant domain refersto a heavy or light chain constant domain. Human IgG heavy chain andlight chain constant domain amino acid sequences are known in the artand disclosed in Table 2 of U.S. Pat. No. 7,915,388, the entire contentsof which are incorporated herein by reference.

Still further, an antibody or antigen-binding portion thereof may bepart of a larger immunoadhesion molecules, formed by covalent ornoncovalent association of the antibody or antibody portion with one ormore other proteins or peptides. Examples of such immunoadhesionmolecules include use of the streptavidin core region to make atetrameric scFv molecule (Kipriyanov et al., Human Antibodies andHybridomas 6:93-101 (1995)) and use of a cysteine residue, a markerpeptide and a C-terminal polyhistidine tag to make bivalent andbiotinylated scFv molecules (Kipriyanov et al., Mol. Immunol.31:1047-1058 (1994)). Antibody portions, such as Fab and F(ab′)₂fragments, can be prepared from whole antibodies using conventionaltechniques, such as papain or pepsin digestion, respectively, of wholeantibodies. Moreover, antibodies, antibody portions and immunoadhesionmolecules can be obtained using standard recombinant DNA techniques, asdescribed herein.

An “isolated antibody”, as used herein, is intended to refer to anantibody that is substantially free of other antibodies having differentantigenic specificities (e.g., an isolated antibody that specificallybinds CD3 is substantially free of antibodies that specifically bindantigens other than CD3). An isolated antibody that specifically bindsCD3 may, however, have cross-reactivity to other antigens, such as CD3molecules from other species. Moreover, an isolated antibody may besubstantially free of other cellular material and/or chemicals.

The term “human antibody”, as used herein, is intended to includeantibodies having variable and constant regions derived from humangermline immunoglobulin sequences. The human antibodies of thedisclosure may include amino acid residues not encoded by human germlineimmunoglobulin sequences (e.g., mutations introduced by random orsite-specific mutagenesis in vitro or by somatic mutation in vivo), forexample in the CDRs and in particular CDR3. However, the term “humanantibody”, as used herein, is not intended to include antibodies inwhich CDR sequences derived from the germline of another mammalianspecies, such as a mouse, have been grafted onto human frameworksequences.

The term “recombinant human antibody”, as used herein, is intended toinclude all human antibodies that are prepared, expressed, created orisolated by recombinant means, such as antibodies expressed using arecombinant expression vector transfected into a host cell (describedfurther in U.S. Pat. No. 7,915,388, the contents of which areincorporated herein by reference), antibodies isolated from arecombinant, combinatorial human antibody library (Hoogenboom et al.,TIB Tech. 15:62-70 (1994); Azzazy et al., Clin. Biochem. 35:425-445(2002); Gavilondo et al., BioTechniques 29:128-145 (2002); Hoogenboom etal., Immunology Today 21:371-378 (2000)), antibodies isolated from ananimal (e.g., a mouse) that is transgenic for human immunoglobulin genes(see e.g., Taylor et al., Nucl. Acids Res. 20:6287-6295 (1992);Kellermann et al., Current Opinion in Biotechnology 13:593-597 (2002);Little et al., Immunology Today 21:364-370 (2002)) or antibodiesprepared, expressed, created or isolated by any other means thatinvolves splicing of human immunoglobulin gene sequences to other DNAsequences. Such recombinant human antibodies have variable and constantregions derived from human germline immunoglobulin sequences. In certainembodiments, however, such recombinant human antibodies are subjected toin vitro mutagenesis (or, when an animal transgenic for human Igsequences is used, in vivo somatic mutagenesis) and thus the amino acidsequences of the VH and VL regions of the recombinant antibodies aresequences that, while derived from and related to human germline VH andVL sequences, may not naturally exist within the human antibody germlinerepertoire in vivo. One embodiment provides fully human antibodiescapable of binding human CD3 which can be generated using techniqueswell known in the art, such as, but not limited to, using human Ig phagelibraries such as those disclosed in Jermutus et al., PCT publicationNo. WO 2005/007699 A2.

The term “chimeric antibody” refers to antibodies which comprise heavyand light chain variable region sequences from one species and constantregion sequences from another species, such as antibodies having murineheavy and light chain variable regions linked to human constant regions.Methods for producing chimeric antibodies are known in the art anddiscussed in to detail in Example 2.1. See e.g., Morrison, Science229:1202 (1985); Oi et al., BioTechniques 4:214 (1986); Gillies et al.,(1989) J. Immunol. Methods 125:191-202; U.S. Pat. Nos. 5,807,715;4,816,567; and 4,816,397, which are incorporated herein by reference intheir entireties. In addition, “chimeric antibodies” may be produced byart-known techniques. See, Morrison et al., 1984, Proc. Natl. Acad. Sci.81:851-855; Neuberger et al., 1984, Nature 312:604-608; Takeda et al.,1985, Nature 314:452-454 which are incorporated herein by reference intheir entireties.

The terms “specific binding” or “specifically binding”, as used herein,in reference to the interaction of an antibody, a protein, or a peptidewith a second chemical species, mean that the interaction is dependentupon the presence of a particular structure (e.g., an antigenicdeterminant or epitope) on the chemical species; for example, anantibody recognizes and binds to a specific protein structure ratherthan to proteins generally. If an antibody is specific for epitope “A”,the presence of a molecule containing epitope A (or free, unlabeled A),in a reaction containing labeled “A” and the antibody, will reduce theamount of labeled A bound to the antibody.

The antibodies used in the scaffolds of the present invention may be“monospecific,” “bi-specific,” or “multispecific.” As used herein, theexpression “antibody” herein is intended to include both monospecificantibodies (e.g., anti-CD3 antibody) as well as bispecific antibodiescomprising an arm that binds to an antigen of interest (e.g., aCD3-binding arm) and a second arm that binds a second target antigen.The target antigen that the other arm of the CD3 bispecific antibodybinds can be any antigen expressed on or in the vicinity of a cell,tissue, organ, microorganism or virus, against which a targeted immuneresponse is desired. In certain embodiments, the CD3-binding arm bindshuman CD3 and induces human T cell proliferation. Also included withinthe meaning of the term are antibodies which bind to different regionsof the CD3 molecule, e.g., an arm that binds to a 17-19 kDa F-chain ofCD3 within the CD3 antigen/T cell antigen receptor (TCR) complex (e.g.,derived from HIT3a), and arm that binds to a 20 kDa subunit of the TCRcomplex within CD3e (e.g., derived from UCHT1). Preferably, the anti-CD3antibody is OKT3 or a CD3-binding fragment thereof.

In one embodiment, the antibody molecule used in the scaffolds of theinvention is a bispecific antibody. Bispecific antibodies may beemployed in the context of the invention to bring a cell of interest,e.g., a cancer cell or a pathogen, in close proximity with the targeteffector cell of the invention, e.g., a cytotoxic T-cell, such that theeffector function of the target effector cell is mediated specificallyupon the cell of interest. Thus, in one embodiment, the inventionprovides scaffolds containing bispecific antibodies, wherein one arm ofthe antibody binds CD3 and the other arm binds a target antigen which isa tumor-associated antigen. Non-limiting examples of specifictumor-associated antigens include, e.g., AFP, ALK, BAGE proteins,β-catenin, brc-abl, BRCA1, BORIS, CA9, carbonic anhydrase IX, caspase-8,CCR5, CD19, CD20, CD30, CD40, CDK4, CEA, CTLA4, cyclin-B1, CYP1 B1,EGFR, EGFRvIII, ErbB2/Her2, ErbB3, ErbB4, ETV6-AML, EpCAM, EphA2, Fra-1,FOLR1, GAGE proteins (e.g., GAGE-1, -2), GD2, GD3, GloboH, glypican-3,GM3, gp100, Her2, HLA/B-raf, HLA/k-ras, H LA/MAG E-A3, hTERT, LMP2, MAGEproteins (e.g., MAGE-1, -2, -3, -4, -6, and -12), MART-1, mesothelin,ML-IAP, Mud, Muc2, Muc3, Muc4, Muc5, Muc16 (CA-125), MUM1, NA17, NY-BR1,NY-BR62, NY-BR85, NY-ESO1, OX40, p15, p53, PAP, PAX3, PAX5, PCTA-1,PLAC1, PRLR, PRAME, PSMA (FOLHI), RAGE proteins, Ras, RGS5, Rho, SART-1,SART-3, Steap-1, Steap-2, survivin, TAG-72, TGF-β, TMPRSS2, Tn, TRP-1,TRP-2, tyrosinase, and uroplakin-3.

In one specific embodiment, the cancer antigen is a member of theepidermal growth factor receptor (EGFR) family, e.g., a receptorselected from the group consisting of EGFR (ErbB-1), HER2/c-neu(ErbB-2), Her 3 (ErbB-3) and Her 4 (ErbB-4), or a mutant thereof.

In another embodiment, the invention relates to scaffolds containing abispecific T-cell engager (BiTE) molecule. The BiTE molecule isspecifically an antibody that recognizes at least one of theaforementioned tumor antigens and at least one T-cell cell surfacemolecule, e.g., CD3. Representative examples of such bispecific T-cellengager molecules include, but are not limited to, solitomab(CD3xEpCAM), blinatumomab (CD3xCD19), MAB MT-111 (CD3xCEA), andBAY-2010112 (CD3xPSMA).

Bispecific antibodies may also be used in the context of the inventionto target effector cells such as T-cells or B-cells to mediate effect onpathogens, e.g., bacteria, viruses, fungus, protists, and othermicrobes, either directly or indirectly. In one embodiment, the pathogenis a virus. In another embodiment, the pathogen is a bacterium.Bispecific antibodies have been used to treat bacterial infections,e.g., drug resistant Pseudomonas aeruginosa. See, DiGiandomenico et al.,Sci Transl Med., 6(262), 2014; Kingwell et al., Nat Rev Drug Discov.,14(1):15, 2015. Other bispecific have been developed to redirectcytotoxic T lymphocytes to kill HIV (Berg et al., Proc Natl Acad Sci.,88(11):4723-7, 1991), protect against HBV infection (Park et al., MolImmunol., 37(18):1123-30, 2000), and other prototypical pathogens(Taylor et al., J Immunol., 159(8):4035-44, 1997).

Accordingly, in one embodiment, the invention provides scaffoldscontaining bispecific antibodies, wherein one arm of the antibody bindsCD3 and the other arm binds a target antigen which is an infectiousdisease-associated antigen (e.g., a bacterial, protozoal, viral, orfungal antigen). Non-limiting examples of infectious disease-associatedantigens include, e.g., an antigen that is expressed on the surface of avirus particle, or preferentially expressed on a cell that is infectedwith a virus, wherein the virus is selected from the group consisting ofHIV, hepatitis (A, B or C), herpes virus (e.g., HSV-1, HSV-2, CMV,HAV-6, VZV, Epstein Barr virus), adenovirus, influenza virus,flavivirus, echovirus, rhinovirus, coxsackie virus, coronavirus,respiratory syncytial virus, mumps virus, rotavirus, measles virus,rubella virus, parvovirus, vaccinia virus, HTLV, dengue virus,papillomavirus, molluscum virus, poliovirus, rabies virus, JC virus, andarboviral encephalitis virus. Alternatively, the target antigen can bean antigen that is expressed on the surface of a bacterium, orpreferentially expressed on a cell that is infected with a bacterium,wherein the bacterium is from a genus selected from the group consistingof Chlamydia, Rickettsia, Mycobacteria, Staphylococci, Streptococci,Pneumonococci, Meningococci, Gonococci, Klebsiella, Proteus, Serratia,Pseudomonas, Legionella, Diphtheria, Salmonella, Bacilli, Clostridium,and Leptospira. In some embodiments, the bacterium causes cholera,tetanus, botulism, anthrax, plague, or Lyme disease. In certainembodiments, the target antigen is an antigen that is expressed on thesurface of a fungus, or preferentially expressed on a cell that isinfected with a fungus, wherein the fungus is selected from the groupconsisting of Candida (e.g., C. albicans, C. krusei, C. glabrata, C.tropicalis, etc.), Crytococcus neoformans, Aspergillus (e.g., A.fumigatus, A. niger, etc.), Mucorales (e.g., M. mucor, M. absidia, M.rhizopus, etc.), Sporothrix schenkii, Blastomyces dermatitidis,Paracoccidioides brasiliensis, Coccidioides immitis, and Histoplasmacapsulatum. In certain embodiments, the target antigen is an antigenthat is expressed on the surface of a parasite, or preferentiallyexpressed on a cell that is infected with a parasite, wherein theparasite is selected from the group consisting of Entamoeba histolytica,Balantidium coli, Naegleriafowleri, Acanthamoeba sp., Giardia lambia,Cryptosporidium sp., Pneumocystis carinii, Plasmodium vivax, Babesiamicroti, Trypanosoma brucei, Trypanosoma cruzi, Leishmania donovani,Toxoplasma gondii, Nippostrongylus brasiliensis, Taenia crassiceps, andBrugia malayi. Non-limiting examples of specific pathogen-associatedantigens include, e.g., HIV gp120, HIV CD4, hepatitis B glycoprotein L,hepatitis B glycoprotein M, hepatitis B glycoprotein S, hepatitis C E1,hepatitis C E2, hepatocyte-specific protein, herpes simplex virus gB,cytomegalovirus gB, and HTLV envelope protein.

In some embodiments, the scaffold of the invention may be used for thetreatment and/or prevention of an allergic reaction or allergicresponse. For example, in some embodiments the scaffold may be used togenerate T-cells (e.g., Tregs) that suppress an allergic response orreaction. For example, in some embodiments, the scaffolds comprise ananti-CD3 antibody and TGF-β. in some embodiments, the scaffolds comprisean anti-CD3 antibody and IL-10. in some embodiments, the scaffoldscomprise an anti-CD3 antibody and rapamycin. In some embodiments, thescaffolds comprise an anti-CD3 antibody, TGF-β, IL-10 and rapamycin. Insome embodiments, the scaffolds comprise an anti-CD3 antibody TGF-β, andIL-10. In some embodiments, the scaffolds comprise an anti-CD3 antibodyand TGF-β and rapamycin. In some embodiments, the scaffolds comprise ananti-CD3 antibody and IL-10 and rapamycin.

In some embodiments, the scaffold of the invention may be used toselectively expand allergen reactive T-cells (e.g., Tregs). In someembodiments the scaffold comprises a peptide derived from an allergen.In some embodiments, the peptide derived from an allergen is presentedon (e.g., complexed with) an MHC molecule (e.g., an MHC class I or MHCclass II molecule). In some embodiments, the MHC molecule is a monomer.In some embodiments the allergen is a food allergen (e.g., a banana,milk, legumes, shellfish, tree nut, stone fruit, egg, fish, soy, orwheat allergen). In one embodiment, the allergen is selected from thegroup consisting of a food allergen, a plant allergen, an insectallergen, an animal allergen, a fungal allergen, a viral allergen, alatex allergen, and a mold spore allergen. In one embodiment, theallergen polypeptide is an insect allergen. In one embodiment, theinsect allergen is a dust mite allergen (e.g., an allergen fromDermatophagoides farina or Dermatophagoides pteronyssinus). In oneembodiment, the allergen polypeptide is an ovalbumin polypeptide. In oneembodiment, the allergen polypeptide is a food allergen polypeptide. Insome embodiments, the scaffold comprises a peptide derived from anallergen and a Th1-skewing cytokine (e.g., IL-12 or IFNγ). In oneembodiment, the allergen polypeptide is a food allergen polypeptide. Insome embodiments, the scaffold comprises a peptide derived from anallergen presented on an MHC molecule and a Th1-skewing cytokine (e.g.,IL-12 or IFNγ). According to certain exemplary embodiments, the presentinvention includes bispecific antigen-binding molecules thatspecifically bind CD3 and CD28. Such molecules may be referred to hereinas, e.g., “anti-CD3/anti-CD28,” or “anti-CD3xCD28” or “CD3xCD28”bispecific molecules, or other similar terminology.

The term “CD28,” as used herein, refers to the human CD28 protein unlessspecified as being from a non-human species (e.g., “mouse CD28,” “monkeyCD28,” etc.). The human CD28 protein has the amino acid sequence shownin GENBANK accession Nos. NP_001230006.1, NP_001230007.1, orNP_006130.1. The mouse CD28 protein has the amino acid sequence shown inGENBANK accession No. NP_031668.3. The various polypeptide sequencesencompassed by the aforementioned accession numbers, include, thecorresponding mRNA and gene sequences, are incorporated by referenceherein in their entirety. As used herein, the expression“antigen-binding molecule” means a protein, polypeptide or molecularcomplex comprising or consisting of at least one complementaritydetermining region (CDR) that alone, or in combination with one or moreadditional CDRs and/or framework regions (FRs), specifically binds to aparticular antigen. In certain embodiments, an antigen-binding moleculeis an antibody or a fragment of an antibody, as those terms are definedelsewhere herein.

As used herein, the expression “bispecific antigen-binding molecule”means a protein, polypeptide or molecular complex comprising at least afirst antigen-binding domain and a second antigen-binding domain. Eachantigen-binding domain within the bispecific antigen-binding moleculecomprises at least one CDR that alone, or in combination with one ormore additional CDRs and/or FRs, specifically binds to a particularantigen. In the context of the present invention, the firstantigen-binding domain specifically binds a first antigen (e.g., CD3),and the second antigen-binding domain specifically binds a second,distinct antigen (e.g., CD28).

The first antigen-binding domain and the second antigen-binding domainof the bispecific antibodies may be directly or indirectly connected toone another. Alternatively, the first antigen-binding domain and thesecond antigen-binding domain may each be connected to a separatemultimerizing domain. The association of one multimerizing domain withanother multimerizing domain facilitates the association between the twoantigen-binding domains, thereby forming a bispecific antigen-bindingmolecule. As used herein, a “multimerizing domain” is any macromolecule,protein, polypeptide, peptide, or amino acid that has the ability toassociate with a second multimerizing domain of the same or similarstructure or constitution. For example, a multimerizing domain may be apolypeptide comprising an immunoglobulin CH3 domain. A non-limitingexample of a multimerizing component is an Fc portion of animmunoglobulin (comprising a CH2-CH3 domain), e.g., an Fc domain of anIgG selected from the isotypes IgG1, IgG2, IgG3, and IgG4, as well asany allotype within each isotype group.

Bispecific antigen-binding molecules of the present invention willtypically comprise two multimerizing domains, e.g., two Fc domains thatare each individually part of a separate antibody heavy chain. The firstand second multimerizing domains may be of the same IgG isotype such as,e.g., lgG1/lgG1, lgG2/lgG2, lgG4/lgG4. Alternatively, the first andsecond multimerizing domains may be of different IgG isotypes such as,e.g., lgG1/lgG2, lgG1/lgG4, lgG2/lgG4, etc.

In certain embodiments, the multimerizing domain is an Fc fragment or anamino acid sequence of 1 to about 200 amino acids in length containingat least one cysteine residues. In other embodiments, the multimerizingdomain is a cysteine residue, or a short cysteine-containing peptide.Other multimerizing domains include peptides or polypeptides comprisingor consisting of a leucine zipper, a helix-loop motif, or a coiled-coilmotif.

Any bispecific antibody format or technology may be used to make thebispecific antigen-binding molecules of the present invention. Forexample, an antibody or fragment thereof having a first antigen bindingspecificity can be functionally linked (e.g., by chemical coupling,genetic fusion, noncovalent association or otherwise) to one or moreother molecular entities, such as another antibody or antibody fragmenthaving a second antigen-binding specificity to produce a bispecificantigen-binding molecule. Specific exemplary bispecific formats that canbe used in the context of the present invention include, withoutlimitation, e.g., scFv-based or diabody bispecific formats, IgG-scFvfusions, dual variable domain (DVD)-lg, Quadroma, knobs-into-holes,common light chain (e.g., common light chain with knobs-into-holes,etc.), CrossMab, CrossFab, (SEED) body, leucine zipper, Duobody,lgG1/lgG2, dual acting Fab (DAF)-lgG, and Mab2 bispecific formats (see,e.g., Klein et al. mAbs 4:6, 1-11, 2012 and references cited therein,for a review of the foregoing formats).

Multispecific antibodies may be specific for different epitopes of onetarget polypeptide or may contain antigen-binding domains specific formore than one target polypeptide. See, e.g., Tutt et al., 1991, J.Immunol. 147:60-69; Kufer et al., 2004, Trends Biotechnol. 22:238-244.The anti-CD3 antibodies of the present invention can be linked to orco-expressed with another functional molecule, e.g., another peptide orprotein. For example, an antibody or fragment thereof can befunctionally linked (e.g., by chemical coupling, genetic fusion,noncovalent association or otherwise) to one or more other molecularentities, such as another antibody or antibody fragment to produce abi-specific or a multispecific antibody with a second bindingspecificity. A multispecific antigen-binding fragment of an antibodywill typically comprise at least two different variable domains, whereineach variable domain is capable of specifically binding to a separateantigen or to a different epitope on the same antigen. Any multispecificantibody format, including the exemplary bispecific antibody formatsdisclosed herein, may be adapted for use in the context of anantigen-binding fragment of an antibody of the present invention usingroutine techniques available in the art. the multispecificantigen-binding molecules of the invention are derived from chimeric,humanized or fully human antibodies. Methods for making multispecicantibodies are well known in the art. For example, one or more of theheavy and/or light chains of the bispecific antigen-binding molecules ofthe present invention can be prepared using VELOCIMMUNE™ technology.Using VELOCIMMUNE™ technology (or any other human antibody generatingtechnology), high affinity chimeric antibodies to a particular antigen(e.g., CD3 or CD28) are initially isolated having a human variableregion and a mouse constant region. The antibodies are characterized andselected for desirable characteristics, including affinity, selectivity,epitope, etc. The mouse constant regions are replaced with a desiredhuman constant region to generate fully human heavy and/or light chainsthat can be incorporated into the bispecific antigen-binding moleculesof the present invention.

In the context of bispecific antigen-binding molecules of the presentinvention, the multimerizing domains, e.g., Fc domains, may comprise oneor more amino acid changes (e.g., insertions, deletions orsubstitutions) as compared to the wild-type, naturally occurring versionof the Fc domain. For example, the invention includes bispecificantigen-binding molecules comprising one or more modifications in the Fcdomain that results in a modified Fc domain having a modified bindinginteraction (e.g., enhanced or diminished) between Fc and FcRn. In oneembodiment, the bispecific antigen-binding molecule comprises amodification in a CH2 or a CH3 region, wherein the modificationincreases the affinity of the Fc domain to FcRn in an acidic environment(e.g., in an endosome where pH ranges from about 5.5 to about 6.0).Non-limiting examples are provided in, for example, US Publication No.2014-0088295. The present invention also includes bispecificantigen-binding molecules comprising a first CH3 domain and a second IgCH3 domain, wherein the first and second Ig CH3 domains differ from oneanother by at least one amino acid, and wherein at least one amino aciddifference reduces binding of the bispecific antibody to Protein A ascompared to a bi-specific antibody lacking the amino acid difference. Incertain embodiments, the Fc domain may be chimeric, combining Fcsequences derived from more than one immunoglobulin isotype.

In another embodiment, the T-cell activating molecule is a majorhistocompatibility complex (MHC) molecule which binds to CD3.Representative examples include, but are not limited to, MHC type Iwhich binds to TCR and CD8 or MHC type II which binds to TCR and CD4.The MHC molecules may be optionally loaded with antigens, e.g.,biotinylated peptides. In other embodiments, the MHC molecules may beconjugated to immunoglobulins, e.g., Fc portion of an immunoglobulin G(IgG) chain. In another embodiment, a plurality of MHC-peptide complexesmay be employed. In the latter case, multiple copies of MHC-peptidecomplexes may be attached, covalently or non-covalently, tomultimerization domains. Known examples of such MHC multimers include,but are not limited to, MHC-dimers (contains two copies of MHC-peptide;IgG is used as multimerization domain, and one of the domains of the MHCprotein is covalently linked to IgG); MHC-tetramers (contains fourcopies of MHC-peptide, each of which is biotinylated and the MHCcomplexes are held together in a complex by the streptavidin tetramerprotein, providing a non-covalent linkage between a streptavidin monomerand the MHC protein); MHC pentamers (contains five copies of MHC-peptidecomplexes are multimerised by a self-assembling coiled-coil domain), MHCdextramers (typically contains more than ten MHC complexes which areattached to a dextran polymer) and MHC streptamers (contains 8-12MHC-peptide complexes attached to streptactin). MHC tetramers aredescribed in U.S. Pat. No. 5,635,363; MHC pentamers are described in theUS patent 2004209295; MHC-dextramers are described in the patentapplication WO 02/072631. MHC streptamers are described in Knabel M etal., Nature Medicine 6. 631-637, 2002).

The target T-cells may also be activated in a CD3-independent manner,for example, via binding and/or ligation of one or more cell-surfacereceptors other than CD3. Representative examples of such cell-surfacemolecules include, e.g., CD2, CD47, CD81, MSR1, etc.

In this context, CD2 is found on virtually all T cells (and also naturalkiller (NK) cells) and is important in T-lymphocyte function. CD2 isassociated with several proteins including CD3, CD5 and CD45. CD2-CD58interaction facilitates cell-cell contact between T cells and APC,thereby enhancing antigen recognition through the TCR/CD3 complex. CD2also serves a signal transduction role. Co-stimulation blockade usingantibodies directed against CD2 may be a potent immunosuppressivestrategy in organ transplantation. Thus, in one embodiment, the T-cellsare activated via the use of an antibody or an antigen binding fragmentthereof that specifically binds to CD2. Representative examples ofanti-CD2 antibodies include, for example, siplizumab (MEDI-507) andLO-CD2b (ATCC accession No. PTA-802; deposited Jun. 22, 1999).

CD47 (IAP) belongs to the immunoglobulin superfamily and partners withmembrane integrins and also binds the ligands thrombospondin-1 (TSP-1)and signal-regulatory protein alpha (SIRPα). See Barclay et al., Curr.Opin. Immunol. 21 (1): 47-52, 2009; Br. J. Pharmacol., 167 (7): 1415-30,2012. CD47 interacts with signal-regulatory protein alpha (SIRPα), aninhibitory transmembrane receptor present on myeloid cells. TheCD47/SIRPα interaction leads to bidirectional signaling, resulting indifferent cell-to-cell responses including inhibition of phagocytosis,stimulation of cell-cell fusion, and T-cell activation. See, Reinhold etal., J Exp Med., 185(1): 1-12, 1997. In accordance with the presentinvention, in one embodiment, the T-cells are activated via the use ofan antibody or an antigen binding fragment thereof that specificallybinds to CD47. Representative examples of anti-CD47 antibodies include,for example, monoclonal antibody Hu5F9-G4, which is being investigatedin various clinical trials against myeloid leukemia and monoclonalantibodies MABL-1 and MABL-2 (FERM Deposit Nos. BP-6100 and BP-6101).See, e.g., WO1999/12973, the disclosure in which is incorporated byreference herein.

CD81 is a member of the tetraspanin superfamily of proteins. It isexpressed on a broad array of tissues, including T cells andhematopoietic cells. CD81 is known to play an immunomodulatory role. Inparticular, cross-linking of CD81 enhances CD3 mediated activation of αβand γδ T-lymphocytes and induces TCR-independent production of cytokinesby γδ T cells in vitro. In accordance with the present invention, in oneembodiment, the T-cells are activated via the use of an antibody or anantigen binding fragment thereof that specifically binds to CD81. See,Menno et al., J. Clin. Invest., 4:1265, 2010. Representative examples ofanti-CD81 antibodies include, for example, monoclonal antibody 5A6. See,e.g., Maecker et al., BMC Immunol., 4:1, 2003, the disclosure in whichis incorporated by reference herein.

MSR1 (CD204) belongs to the family of class A macrophage scavengerreceptors, which include three different types (1, 2, 3) generated byalternative splicing of the MSR1 gene. These receptors or isoforms aretrimeric integral membrane glycoproteins and have been implicated inmany macrophage-associated physiological and pathological processesincluding atherosclerosis, Alzheimer's disease, and host defense. See,Matsumoto et al., Proc. Natl. Acad. Sci. U.S.A. 87 (23): 9133-7, 1990.Recent studies demonstrate that dendritic (DC) MSR1 impacts theactivation and proliferation of CD8 T cells and antibody-mediatedblocking of MSR1 increased proliferation and expansion of T-cells invitro. Lerret et al., PLoS One., 7(7):e41240, 2012. In accordance withthe present invention, in one embodiment, the T-cells are activated viathe use of an antibody or an antigen binding fragment thereof thatspecifically binds to MSR1. Representative examples of anti-MSR1antibodies include, for example, rat anti-human CD204 antibody (ThermoCatalog No. MA5-16494) and goat anti-human CD204/MSR1 antibody (BioradCatalog No. AHP563).

In another embodiment, the T-cells are activated by ligating/binding toa T-cell receptor (TCR) molecule, which is expressed ubiquitously inT-cells. The TCR is a heterodimer composed of two different proteinchains. In humans, in 95% of T cells the TCR consists of an alpha (a)and beta (β) chain, whereas in 5% of T cells the TCR consists of gammaand delta (γ/δ) chains. When the TCR engages with antigenic peptide andMHC (peptide/MHC), the T lymphocyte is activated through signaltransduction. In accordance with the present invention, in oneembodiment, the T-cells are activated via the use of an antibody or anantigen binding fragment thereof that specifically binds to TCR.Representative examples of anti-TCR antibodies include, for example,mouse anti-human TCR monoclonal antibody IMMU510 (Immunotech, BeckmanCoulter, Fullerton, CA)(described in Zhou et al., Cell Mol Immunol.,9(1): 34-44, 2012) and monoclonal antibody defining alpha/beta TCR WT31(described in Gupta et al., Cell Immunol., 132(1):26-44, 1991).

In another embodiment, the T-cell activating molecule is a majorhistocompatibility complex (MHC) molecule that is optionally loaded withan MHC peptide. There are two general classes of MHC molecules. Class IMHC (pMHC) molecules are found on almost all cells and present peptidesto cytotoxic T lymphocytes (CTL). Class II MHC molecules are foundmainly on antigen-presenting immune cells (APCs), which ingestpolypeptide antigens (in, for example, microbes) and digest them intopeptide fragments. The MHC-II molecules then present the peptidefragments to helper T cells, which, after activation, provide generallyrequired helper activity for responses of other cells of the immunesystem (e.g., CTL or antibody-producing B cells). The interactionbetween the peptide bound in the binding cleft of the heavy chain of MHCclass I (pMHC) and the complementary determining regions (CDR) of the Tcell receptor (TCR) determines the potential for T cell activationduring the afferent and efferent stages of cellular immunity. Theaffinity that exists between TCR and MHC-peptide complex regulates Tcell fate during development, initial activation, and during executionof effector functions.

Accordingly, in one embodiment, the instant invention relates to MSR-SLBscaffolds containing a human MHC molecule optionally loaded with apeptide. Representative examples of such MHC molecules include HLA-A,HLA-B, HLA-C, DP, DQ and DR, or a combination thereof.

The MHC molecules may be monovalent or bivalent. In some embodiments,bivalency or multivalency of the MHC molecules is desirable for signaldelivery (either activation or inhibition signals) to the T cell.Therefore, in some embodiments, the MSR-SLB scaffolds of the presentinvention include at least two identical MHC molecules attached to alinker.

The linker of the bivalent MHC molecule serves three functions. First,the linker contributes the required bivalency or multivalency. Second,the linker increases the half-life of the entire fusion protein in vivo.Third, the linker determines whether the fusion protein will activate orsuppress T cells. T cell priming requires stimulation via the TCR and anadditional second signal generally delivered by the APC. In the absenceof a second signal, T cell hyporesponsiveness may result. Byconstructing a fusion protein in which the linker allows delivery of asecond signal, T cell stimulation results in enhanced T cell immunity.By constructing a fusion protein in which the linker does not providefor delivery of a second signal, T cell suppression results inimmunosuppression. A fusion protein with T cell stimulatory propertiescan be constructed by using a linker which allows for delivery of asecond signal to the T cell in addition to the signal delivered via theTCR. This can be accomplished by using a linker that has bindingaffinity for a cell surface structure on another cell, that cell beingcapable of delivering a second signal to the T cell. Thus, the linkerserves to bridge the T cell and the other cell. By bringing the othercell into close proximity to the T cell, the other cell can deliver asecond signal to the T cell.

Examples include linkers that can bind to Fc receptors on other cellssuch as certain immunoglobulin chains or portions of immunoglobulinchains. Specific examples include IgG, IgA, IgD, IgE, and IgM. When animmunoglobulin is used, the entire protein is not required. For example,the immunoglobulin gene can be cleaved at the hinge region and only thegene encoding the hinge, CH2, and CH3 domains of the heavy chain is usedto form the fusion protein. The linker may bind other cell surfacestructures. For example, the linker can include a cognate moiety formany cell surface antigens which can serve as a bridge to bring thesecond cell into close proximity with the T cell. The linker might alsodeliver a second signal independently. For example, a linker withbinding affinity for the T cell antigen CD28 can deliver a secondsignal. In addition, the linker can increase the half-life of the entirefusion protein in vivo. A fusion protein with T cell inhibitoryproperties can be constructed by using a linker that does not result indelivery of a second signal. Examples include Ig chains that do not bindFc receptor, Ig F(ab′)2 fragments, a zinc finger motif, a leucinezipper, and non-biological materials. Examples of non-biologicalmaterials include plastic microbeads, or even a larger plastic membersuch as a plastic rod or tube, as well as other physiologicallyacceptable carriers which are implantable in vivo.

In some embodiments, the MHC molecules are not attached to a linker.Without wishing to be bound by any particular theory, it is believedthat the fluid nature of the lipid bilayer allows T cells to reorganizethe membrane and form multivalent clusters. These clusters cansubsequently be disassembled, which would not be possible if thesignaling molecules were attached together with a linker. Inability toun-form these multivalent clusters can potentially lead tooverstimulation and T cell exhaustion or anergy (see, e.g., Lee K-H etal. Science 302(5648): 1218-22 (2003)).

In some embodiments, the lipid bilayer of the APC-MS comprises a lipidcomposition that favor the spontaneous partitioning of lipid speciesinto liquid-ordered domains (see, e.g., Wang T-Y et al. Biochemistry40(43):13031-40 (2001)).

Optionally, the MHC molecules may be loaded with a specific peptide(e.g., a peptide derived from a viral antigen, a bacterial antigen, oran allergen). The specific peptide of the fusion protein can be loadedinto the MHC molecules after the fusion protein has been made. Thepeptide may also be subsequently covalently attached to the MHC, forexample by UV cross-linking. Alternatively, a peptide sequence can beincorporated into the DNA sequence encoding the fusion protein such thatthe peptide is loaded into the MHC molecules during generation of thefusion protein. In the latter case, the peptide can be attached with atether, such as polylysine, which allows it to complex with the MHCportion of the fusion protein. The specific peptides to be loaded intothe MHC molecules are virtually limitless and are determined based onthe desired application. For example, to enhance T cell immunity,peptides from various sources, e.g., viral, fungal and bacterialinfections, or to tumors, can be used. To suppress T cell immunity inautoimmunity, autoreactive peptides can be used. To suppress T cellimmunity to transplanted tissues, self-peptides which are presented byalloantigens can be used.

Toxins, such as ricin and diphtheria toxin, and radioisotopes, may becomplexed to the fusion protein (for example, using5-methyl-2-iminothiolane) to kill the specific T cell clones. Thesetoxins can be chemically coupled to the linker or to the MHC portion ofthe fusion protein, or they can be incorporated into the DNA sequenceencoding the fusion protein such that the toxin is complexed to thefusion protein during generation of the fusion protein.

The MHC-peptide/immunoglobulin fusion protein can be prepared byconstructing a gene which encodes for the production of the fusionprotein. Alternatively, the components of the fusion protein can beassembled using chemical methods of conjugation. Sources of the genesencoding the MHC molecules and the linkers can be obtained from variousdatabases. In the case of MHC class I fusion proteins, the MHC fragmentcan be attached to the linker and P2 microglobulin can be allowed toself-associate. Alternatively, the fusion protein gene can beconstructed such that P2 microglobulin is attached to the MHC fragmentby a tether. In the case of MHC class II fusion protein, either thealpha or the beta chain can be attached to the linker and the otherchain can be allowed to self-associate. Alternatively, the fusionprotein gene can be constructed such that the alpha and beta chains areconnected by a tether. Peptides can be prepared by encoding them intothe fusion protein gene construct or, alternatively, with peptidesynthesizers using standard methodologies available to one of ordinaryskill in the art. The resultant complete fusion proteins can beadministered using routine techniques.

T-Cell Co-Stimulatory Molecules

In one embodiment, the instant invention provides MSR-SLB scaffoldscontaining a plurality of T-cell co-stimulatory molecules. Theseco-stimulatory molecules may mediate direct, indirect, or semi-directstimulation of a target population of T-cells. Preferably, theco-stimulatory molecules mediate activation of T-cells in the presenceof one or more T-cell activating molecules.

The term “co-stimulatory molecule” is used herein in accordance with itsart recognized meaning in immune T cell activation. Specifically, a“co-stimulatory molecule” refers to a group of immune cell surfacereceptor/ligands which engage between T cells and antigen presentingcells and generate a stimulatory signal in T cells which combines withthe stimulatory signal (i.e., “co-stimulation”) in T cells that resultsfrom T cell receptor (“TCR”) recognition of antigen on antigenpresenting cells. As used herein, a soluble form of a co-stimulatorymolecule “derived from an APC” refers to a co-stimulatory moleculenormally expressed by B cells, macrophages, monocytes, dendritic cellsand other APCs. See, Huppa et al., Nature Reviews Immunology. 3, 973-983(2003). A “co-stimulator of T cells activation” refers to the ability ofa co-stimulatory ligand to bind and to activate T cells which have beenactivated via any of the aforementioned mechanisms or pathways, e.g.,via CD3-dependent or CD3-independent T-cell activation. Co-stimulatoryactivation can be measured for T cells by the production of cytokines asis well known and by proliferation assays that are well known (e.g.,CFSE staining) and/or as described in the examples below.

In one embodiment, the instant invention provides for MSR-SLB scaffoldscontaining molecules that specifically bind to a co-stimulatory antigen.Particularly, the MSR-SLB scaffolds contain a plurality of T-cellcostimulatory molecules which specifically bind to CD28, 4.1BB (CD137),OX40 (CD134), CD27 (TNFRSF7), GITR (CD357), CD30 (TNFRSF8), HVEM(CD270), LTβR (TNFRSF3), DR3 (TNFRSF25), ICOS (CD278), CD226 (DNAM1),CRTAM (CD355), TIM1 (HAVCR1, KIM1), CD2 (LFA2, OX34), SLAM (CD150,SLAMF1), 2B4 (CD244, SLAMF4), Ly108 (NTBA, CD352, SLAMF6), CD84(SLAMF5), Ly9 (CD229, SLAMF3), CD279 (PD-1) and/or CRACC (CD319, BLAME).

In one embodiment, the co-stimulatory molecule is an antibody or anantigen binding fragment thereof which binds specifically to one or moreof the aforementioned co-stimulatory antigens. In this context, CD28 isthe prototypic T cell co-stimulatory antigen and binds to molecules ofthe B7 family expressed on APCs such as dendritic cells and activated Bcells. Human CD28 is found on all CD4+ T cells and on about half of CD8+T cells. T cell activities attributed to CD28 include prevention ofenergy, induction of cytokine gene transcription, stabilization ofcytokine mRNA and activation of CD8+ cytotoxic T lymphocytes. Theligands for CD28 identified as CD80(B7-1) and CD86(B7-2) areimmunoglobulin superfamily monomeric transmembrane glycoproteins of 60kDa and 80 kDa respectively.

In one embodiment, the instant invention relates to MSR-SLB scaffoldscontaining an antibody or an antigen-binding fragment thereof whichbinds specifically to CD28. Representative examples of anti-CD28antibodies include, for example, lulizumab pegol and TGN1412. See alsoU.S. Pat. No. 8,785,604.

In another embodiment, the instant invention relates to MSR-SLBscaffolds containing an antibody or an antigen-binding fragment thereofwhich binds specifically to ICOS (CD278). ICOS is a CD28-superfamilycostimulatory molecule that is expressed on activated T cells. It isthought to be important for Th2 cells in particular. Representativeexamples of anti-ICOS antibodies include, for example, monoclonalantibody 2C7, which recognizes the ICOS molecule expressed on activatedT cells and induces the activation as well as proliferation of T cellsprestimulated by anti-human CD3 monoclonal antibodies. See Deng et al.,Hybrid Hybridomics., 23(3):176-82, 2004.

In another embodiment, the instant invention provides for MSR-SLBscaffolds containing an antibody or an antigen-binding fragment thereofwhich binds specifically to CD152 (CTLA4). The antibody is preferably aneutralizing antibody or a blocking antibody. CD152 is expressed onactivated CD4+ and CD8+ T cells, and on regulatory T-cells (Tregs). Itsfunctions in T-cell biology, during immune responses to infection, andas a target for cancer immunotherapy have been well described (Egen etal., Nat. Immunol., 3(7):611-618, 2002). CTLA-4 is a homologouscounterpart to CD28, both of which bind to CD80 and CD86 on APCs. Theimportance of CTLA-4 for immune tolerance is clear (Waterhouse et al.,Science, 270(5238):985-988, 1995). These include out-competing loweraffinity CD28 molecules for ligand binding to minimize T-cellco-stimulation, recruitment of inhibitory phosphatases to the TCRcomplex to disrupt positive signaling cascades, and removing CD80 andCD86 from the surface of APC by trans-endocytosis, thereby diminishingthe ability of APC to properly activate otherwise responsive T-cells.Accordingly, exploitation of the CTLA-4 receptor/pathway is anattractive strategy to modulate T-cell immunity. Indeed, anti-CTLA-4 wasthe first monoclonal antibody (ipilimumab) to be FDA-approved forcheckpoint blockade treatment in cancer patients. Other examples ofCTLA-4 antibodies that may be employed in accordance with the instantinvention include tremelimumab and antigen-binding fragments thereof.

In another embodiment, the instant invention provides for MSR-SLBscaffolds containing an antibody or an antigen-binding fragment thereofwhich binds specifically to programmed death-1 (PD-1; CD279). PD1 is amember of the same family of receptors as CD28 and CTLA-4, and isbroadly expressed on lymphoid and myeloid cells. PD-1 binds uniquely tothe B7 ligands PD-L1 and PD-L2 on APC and other surrounding tissues,greatly influencing the fate of responding CD8+ T cells in settings ofchronic infections. On T-cells, PD-1 is expressed after antigenencounter, but acts almost immediately to impede T-cell activation byrecruiting the phosphatases SHP-1 and SHP-2 through signaling motifs inthe PD-1 cytoplasmic tail, which reduces Akt phosphorylation, anddiminishes T-cell metabolism, proliferation and survival. Accordingly,the antibody is preferably a neutralizing antibody or a blockingantibody. Representative examples of such anti-PD-1 antibodies include,for example, nivolumab, lambrolizumab (MK-3475), pidilizumab (CT-011)and AMP-224.

In another embodiment, the instant invention relates to MSR-SLBscaffolds containing an antibody or an antigen-binding fragment thereofwhich binds specifically to CD81. Engagement of CD81 lowers thesignaling threshold required to trigger T-Cell/CD3 mediated proviral DNAin CD4+ T cells (Tardif et al., J. Virol. 79 (7): 4316-28, 2005).Representative examples of anti-CD81 antibodies include, for example,monoclonal antibody 5A6. See, e.g., Maecker et al., BMC Immunol., 4:1,2003, the disclosure in which is incorporated by reference herein.

In another embodiment, the instant invention relates to MSR-SLBscaffolds containing an antibody or an antigen-binding fragment thereofwhich binds specifically to CD137. Crosslinking of CD137 enhances T cellproliferation, IL-2 secretion, survival and cytolytic activity. Further,it can enhance immune activity to eliminate tumors in vivo. Accordingly,the antibodies that bind to CD137 are preferably agonistic antibodies.Representative examples of anti-CD137 antibodies include, for example,monoclonal antibody utomilumab, which is a human IgG that is currentlybeing investigated in clinical trials. See National Clinical Trials ID:NCT01307267.

In another embodiment, the instant invention relates to MSR-SLBscaffolds containing an antibody or an antigen-binding fragment thereofwhich binds specifically to OX40 (CD134). OX40L binds to OX40 receptorson T-cells, preventing them from dying and subsequently increasingcytokine production. OX40 has a critical role in the maintenance of animmune response beyond the first few days and onwards to a memoryresponse due to its ability to enhance survival. OX40 also plays acrucial role in both Th1 and Th2 mediated reactions in vivo.Accordingly, the antibodies that bind to OX40 are preferably agonisticantibodies. Representative examples of anti-OX40 antibodies include, forexample, anti-OX40 monoclonal antibody utomilumab, which is beinginvestigated in various clinical trials (see National Clinical TrialsID: NCT01644968, NCT01303705 and NCT01862900).

In another embodiment, the instant invention relates to MSR-SLBscaffolds containing an antibody or an antigen-binding fragment thereofwhich binds specifically to CD27 (TNFRSF7). CD27 a member of theTNF-receptor superfamily and is required for generation and long-termmaintenance of T cell immunity. It binds to ligand CD70, and plays a keyrole in regulating immunoglobulin synthesis. CD27 supportsantigen-specific expansion (but not effector cell maturation) of naïve Tcells, independent of the cell cycle-promoting activities of CD28 andIL2 (Hendriks et al., Nature Immunology 1, 433-440, 2000)). As such, theMSR-SLB scaffolds of the invention preferably include agonisticantibodies that bind to CD27. Representative examples of anti-CD27antibodies include, for example, the monoclonal antibody varlilumab. SeeRamakrishna et al., Journal for ImmunoTherapy of Cancer, 3:37, 2015.

In another embodiment, the instant invention relates to MSR-SLBscaffolds containing an antibody or an antigen-binding fragment thereofwhich binds specifically to glucocorticoid-induced TNF receptorfamily-regulated gene (GITR or CD357). GITR is a 25 kDa TNF receptorsuperfamily member which is expressed on activated lymphocytes. GITR isupregulated by T cell receptor engagement. The cytoplasmic domain ofGITR is homologous to CD40, 4-1BB and CD27. GITR signaling has beenshown to regulate T cell proliferation and TCR-mediated apoptosis, andto break immunological self-tolerance. GITR further binds GITRL and isinvolved in the development of regulatory T cells and to regulate theactivity of Th1 subsets. Modulation of GITR with agonistic antibodieshas been shown to amplify the antitumor immune responses in animalmodels via multiple mechanisms. Anti-GITR antibodies are designed toactivate the GITR receptor thereby increasing the proliferation andfunction of effector T cells. At the same time, ligation of GITR onsurface of Tregs could abrogate suppressive function of these cells ontumor specific effector T-cells thus further augmenting T-cell immuneresponse. Representative examples of anti-GITR antibodies include, forexample, humanized, Fc disabled anti-human GITR monoclonal antibodyTRX518, which induces both the activation of tumor-antigen-specific Teffector cells, as well as abrogating the suppression induced byinappropriately activated T regulatory cells. TRX518 is beinginvestigated in various clinical trials (see National Clinical TrialsID: NCT01239134).

In another embodiment, the instant invention relates to MSR-SLBscaffolds containing an antibody or an antigen-binding fragment thereofwhich binds specifically to CD30 (TNFRSF8). CD30 antigen is atrans-membrane glycoprotein belonging to the tumor necrosis factorreceptor superfamily, which, when stimulated, exerts pleiotropic effectson cell growth and survival. In normal or inflamed tissues, CD30expression is restricted to medium/large activated B and/orT-lymphocytes. It is expressed by activated, but not by resting, T and Bcells (Guo et al., Infect. Immun., 81 (10), 3923-3934, 2013).Stimulation of CD30L/CD30 signaling by in vivo administration ofagonistic anti-CD30 monoclonal antibody (MAb) restored IL-17A productionby Vγ1-Vγ4-γδ T cells in CD30L knockout mice. Representative examples ofanti-CD30 antibodies include, for example, brentuximab vedotin(Adcetris).

In another embodiment, the instant invention relates to MSR-SLBscaffolds containing an antibody or an antigen-binding fragment thereofwhich binds specifically to HVEM (CD270). CD270 is a member of theTNF-receptor superfamily. This receptor was identified as a cellularmediator of herpes simplex virus (HSV) entry. Mutations in this genehave been recurrently been associated to cases of diffuse large B-celllymphoma. Representative examples of anti-CD270 antibodies include, forexample, the monoclonal antibody HVEM-122. See, Cheung et al., J.Immunol., 185:1949, 2010; Hobo et al., J Immunol., 189:39, 2012.

In another embodiment, the instant invention relates to MSR-SLBscaffolds containing an antibody or an antigen-binding fragment thereofwhich binds specifically to lymphotoxin beta receptor (LTβR; TNFRSF3).LTβR is involved in CD4+ T-cell priming (Summers deLuca et al., J ExpMed., 204(5):1071-81, 2007). Representative examples of anti-LTβRantibodies include, for example, the monoclonal antibody BBF6 antibody.See also WO2010/078526, which is incorporated by reference.

In another embodiment, the instant invention relates to MSR-SLBscaffolds containing an antibody or an antigen-binding fragment thereofwhich binds specifically to DR3 (TNFRSF25). DR3 is thought to beinvolved in controlling lymphocyte proliferation induced by T-cellactivation. Specifically, activation of DR3 is dependent upon previousengagement of the T cell receptor. Following binding to TL1A, DR3signaling increases the sensitivity of T cells to endogenous IL-2 viathe IL-2 receptor and enhances T cell proliferation. Because theactivation of the receptor is T cell receptor dependent, the activity ofDR3 in vivo is specific to those T cells that are encountering cognateantigen. At rest, and for individuals without underlying autoimmunity,the majority of T cells that regularly encounter cognate antigen areFoxP3+ regulatory T cells. Stimulation of TNFRSF25, in the absence ofany other exogenous signals, stimulates profound and highly specificproliferation of FoxP3+ regulatory T cells from their 8-10% of all CD4+T cells to 35-40% of all CD4+ T cells within 5 days. Representativeexamples of DR3 agonists include, for example, antibodies bindingspecifically to DR3 (Reddy et al., J. Virol., 86 (19) 10606-10620, 2012)and the agonist 4C12 (Wolf et al., Transplantation, 27; 94(6):569-74,2012).

In another embodiment, the instant invention relates to MSR-SLBscaffolds containing an antibody or an antigen-binding fragment thereofwhich binds specifically to CD226 (DNAM1). CD226 is a ˜65 kDaglycoprotein expressed on the surface of natural killer cells,platelets, monocytes and a subset of T cells. It is a member of theimmunoglobulin superfamily and mediates cellular adhesion to other cellsbearing its ligands, CD 112 and CD155. Cross-linking CD226 withantibodies causes cellular activation and ligation of CD226 and LFA-1with their respective ligands cooperates in triggering cytotoxicity andcytokine secretion by T and NK cells (Tahara et al., Int. Immunol. 16(4): 533-8, 2004).

In another embodiment, the instant invention relates to MSR-SLBscaffolds containing an antibody or an antigen-binding fragment thereofwhich binds specifically to CRTAM (CD355). CTRAM is anMHC-class-I-restricted T-cell-associated molecule, which regulates latephase of cell polarity in some CD4+ T cells. CTRAM also regulatesinterferon-γ (IFNγ) and interleukin-22 (IL-22) production. In oneembodiment, the MSR-SLB scaffolds comprise a monoclonal anti-CTRAMantibody. Representative examples of CTRAM antibodies include, forexample, the mouse anti human CTRAM antibody 21A9 (GENTEX Inc. USA,Irvine, CA).

In another embodiment, the instant invention relates to MSR-SLBscaffolds containing an antibody or an antigen-binding fragment thereofwhich binds specifically to TIM1 (HAVCR1, KIM1). TIM genes belong totype I cell-surface glycoproteins, which include an N-terminalimmunoglobulin (Ig)-like domain, a mucin domain with distinct length, asingle transmembrane domain, and a C-terminal short cytoplasmic tail.The localization and functions of TIM genes are divergent between eachmember. TIM-1 is preferentially expressed on Th2 cells and has beenidentified as a stimulatory molecule for T-cell activation (Umetsu etal., Nat. Immunol. 6 (5): 447-54, 2005). In one embodiment, the MSR-SLBscaffolds comprise a monoclonal anti-TIM1 antibody. Representativeexamples of TIM1 antibodies include, for example, the rabbit anti humanTIM1 antibody ab47635 (ABCAM, Cambridge, MA).

In another embodiment, the instant invention relates to MSR-SLBscaffolds containing an antibody or an antigen-binding fragment thereofwhich binds specifically to SLAM (CD150, SLAMF1). SLAM (CD150) is aself-ligand and cell surface receptor that functions as a costimulatorymolecule and also a microbial sensor that controlled the killing ofGram-negative bacteria by macrophages. In particular, SLAM regulatedactivity of the NADPH oxidase NOX2 complex and phagolysosomal maturationafter entering the phagosome, following interaction with the bacterialouter membrane proteins (Berger et al., Nature Immunology 11, 920-927,2010). Slamf1 is expressed on the surface of activated and memory Tcells as well as on activated B cells, dendritic cells, macrophages andplatelets (Calpe et al., Adv. Immunol. 2008; 97:177). In one embodiment,the MSR-SLB scaffolds comprise a monoclonal anti-SLAM1 antibody or anantigen-binding fragment thereof. Representative examples of SLAM1antibodies include, e.g., the rabbit anti human SLAM1 antibody600-401-EN3 (Rockland Antibodies, Limerick, PA).

In another embodiment, the instant invention relates to MSR-SLBscaffolds containing an antibody or an antigen-binding fragment thereofwhich binds specifically to 2B4 (CD244, SLAMF4). CD244 is a cell surfacereceptor expressed on natural killer cells (NK cells) (and some T cells)mediating non-major histocompatibility complex (MHC) restricted killing.The interaction between NK-cell and target cells via this receptor isthought to modulate NK-cell cytolytic activity. CD244 is a co-inhibitorySLAM family member which attenuates primary antigen-specific CD8(+) Tcell responses in the presence of immune modulation with selective CD28blockade. Recent studies reveal a specific up-regulation of 2B4 onantigen-specific CD8(+) T cells in animals in which CD28 signaling wasblocked (Liu et al., J Exp Med. 2014 Feb. 10; 211(2):297-311). In oneembodiment, the MSR-SLB scaffolds comprise a monoclonal anti-CD244antibody or an antigen-binding fragment thereof. Representative examplesof CD244 antibodies include, e.g., anti-2B4 antibody C1.7 orPE-conjugated anti-2B4 (C1.7), which have been characterized in Sanduskyet al. (Eur J Immunol. 2006 December; 36(12):3268-76).

In another embodiment, the instant invention relates to MSR-SLBscaffolds containing an antibody or an antigen-binding fragment thereofwhich binds specifically to Ly108 (NTBA, CD352, SLAMF6). SLAMF6 is atype I transmembrane protein, belonging to the CD2 subfamily of theimmunoglobulin superfamily, which is expressed on natural killer (NK),T, and B lymphocytes. Co-stimulation of T lymphocytes through theSLAMF3/SLAMF6 pathways mediates more potent effects on IL-17A expressionwhen compared with the canonical CD28 pathway. SLAMF3/SLAMF6 signalingmediates increased nuclear abundance and recruitment of RORγt to theproximal IL17A promoter, resulting in increased trans-activation andgene expression (Chatterjee et al., J Biol Chem., 287(45): 38168-38177,2012). In one embodiment, the MSR-SLB scaffolds comprise a monoclonalanti-CD244 antibody or an antigen-binding fragment thereof.Representative examples of CD244 antibodies include, e.g., anti NTB-Aantibodies characterized in Flaig et al. (J. Immunol. 2004. 172:6524-6527) and Stark et al. (J. Immunol. Methods 2005. 296: 149-158).

In another embodiment, the instant invention relates to MSR-SLBscaffolds containing an antibody or an antigen-binding fragment thereofwhich binds specifically to CD84 (SLAMF5). CD84 is a member of the CD2subgroup of the immunoglobulin receptor superfamily. Members of thisfamily have been implicated in the activation of T cells and NK cells.CD84 increases proliferative responses of activated T-cells andhomophilic interactions enhance interferon gamma secretion inlymphocytes. CD84 may also serve as a marker for hematopoieticprogenitor cells. See the disclosure in the references with the PUBMEDID Nos. 11564780, 12115647, 12928397, 12962726, 16037392, which indicatethat it is required for a prolonged T-cell: B-cell contact, optimal Thfunction, and germinal center formation. In one embodiment, the MSR-SLBscaffolds comprise a monoclonal anti-CD84 antibody or an antigen-bindingfragment thereof. Representative examples of CD84 antibodies include,e.g., PE anti-human CD84 antibody CD84.1.21, which is able to enhanceCD3 induced IFN-γ production and partially block CD84-Ig binding tolymphocytes (BioLegend, San Diego, CA; Catalog No. 326008).

In another embodiment, the instant invention relates to MSR-SLBscaffolds containing an antibody or an antigen-binding fragment thereofwhich binds specifically to Ly9 (CD229, SLAMF3). CD229 participates inadhesion reactions between T lymphocytes and accessory cells byhomophilic interaction. It also promotes T-cell differentiation into ahelper T-cell Th17 phenotype leading to increased IL-17 secretion; thecostimulatory activity requires SH2D1A (Chatterjee et al., J Biol Chem.,287(45): 38168-38177, 2012). In particular, concurrent ligation of CD229and TCR with immobilized CD229-His protein and anti-CD3 antibodysignificantly enhanced cell proliferation and IFN-γ secretion in murineCD3+ splenocytes in a dose-dependent manner (Wang et al., The Journal ofImmunology, 188 (sup. 1) 176.7, May 2012). Accordingly, in oneembodiment, the MSR-SLB scaffolds comprise a monoclonal anti-CD229antibody or an antigen-binding fragment thereof. Representative examplesof CD229 antibodies include, e.g., PE anti-human CD229 antibodyHLy-9.1.25 (BIOLEGEND, San Diego, CA; Catalog No. 326108) or mouseanti-human CD229 antibody (R&D Systems Catalog No. AF1898).

In another embodiment, the instant invention relates to MSR-SLBscaffolds containing an antibody or an antigen-binding fragment thereofwhich binds specifically to CD279 (PD-1). PD-1 functions as an immunecheckpoint and plays an important role in down regulating the immunesystem by preventing the activation of T-cells, which in turn reducesautoimmunity and promotes self-tolerance. The inhibitory effect of PD-1is accomplished through a dual mechanism of promoting apoptosis(programmed cell death) in antigen specific T-cells in lymph nodes whilesimultaneously reducing apoptosis in regulatory T cells (suppressor Tcells). Representative examples of CD229 antibodies include, e.g.,nivolumab, pembrolizumab, pidilizumab (CT-011, Cure Tech), BMS936559,and atezolizumab.

In another embodiment, the instant invention relates to MSR-SLBscaffolds containing an antibody or an antigen-binding fragment thereofwhich binds specifically to CRACC (CD319, BLAME). CD319 mediates NK cellactivation through a SH2D1A-independent extracellular signal-regulatedERK-mediated pathway (Bouchon et al., J Immunol. 2001 Nov. 15;167(10):5517-21). CD319 also positively regulates NK cell functions andmay contribute to the activation of NK cells. Accordingly, in oneembodiment, the MSR-SLB scaffolds comprise a monoclonal anti-CD319antibody or an antigen-binding fragment thereof. Representative examplesof CD319 antibodies include, e.g., elotuzumab or an antigen-bindingfragment thereof.

In certain embodiments, the instant invention provides for MSR-SLBscaffolds containing a binding pair containing at least one T-cellactivating molecule and at least one T-cell co-stimulatory molecule.Representative examples of such pairs include, but are not limited to,for example, antibodies binding to CD3/CD28, CD3/ICOS, CD3/CD27, andCD3/CD137, or a combination thereof. In this context, depending on thedesired modulation of activity of co-stimulatory molecules, it may bedesirable to employ an agonist antibody for the first component (CD3)and an agonist or antagonist antibody for the second component.

In certain embodiments, the instant invention provides for MSR-SLBscaffolds containing a binding pair containing at least one T-cellactivating molecule which is an antibody binding to CD3 and at least oneT-cell co-stimulatory molecule which is an antibody binding to CD28,optionally together with a second co-stimulatory molecule which is anantibody binding to an antigen selected from the group consisting ofICOS, CD27, and CD137. In one embodiment, the MSR-SLB scaffold containsa combination of functional molecules selected from the followingcombinations: (a) antibodies which bind to CD3, CD28 and ICOS, (b)antibodies which bind to CD3, CD28 and CD27, (c) antibodies which bindto CD3, CD28 and CD137, (d) antibodies which bind to CD3, CD28, ICOS andCD27. In this regard, experimental data suggests that stimulation ofthese secondary T-cell co-stimulation factors may stimulatedifferentiation of certain types of T-cells when applied withappropriate activation stimuli such as CD3+CD28. For example, ICOSstimulation favors differentiation of Th effector cells when cooperateswith CD3+CD28+ stimulation, whereas it supports differentiation ofregulatory T cells when costimulatory signals are insufficient. See,Mesturini et al., Eur J Immunol., 36(10):2601-12, 2006. Similarly,anti-CD27 antibodies may be used to fine-tune the system. In thiscontext, anti-CD27 antibody 1F5 (when used together with anti-CD3antibodies) did not trigger potentially dangerous polyclonal T-cellactivation—a phenomena observed with co-stimulatory CD28-specificsuper-agonistic antibodies. See, Thomas et al., Oncoimmunology, 3:e27255, 2014.

In one embodiment, the binding pair includes monospecific antibodies,wherein a first antibody binds to a first member of the pair, e.g., CD3,and a second antibody binds to a second member of the pair, e.g., CD28.In another embodiment, the pair includes bispecific antibodies, whereina single antibody binds to the individual pair members, e.g., abispecific antibody binding to CD3 and CD28. In this context, bispecificantibodies are preferred due to their ability to confer enhanced T-cellactivation. See, Willems et al., Cancer Immunol Immunother. 2005November; 54(11):1059-71.

Alternately, the binding pair includes monospecific antibodies, whereina first antibody binds to CD3 and a second antibody binds to ICOS. Inthe context of the antibody binding to ICOS, insofar as the molecule hasbeen implicated in the etiology of graft-versus-host diseases (see, Satoet al., Transplantation, 96(1): 34-41, 2013), it may be preferable toemploy an antagonistic antibody that neutralizes ICOS. A bispecificantibody containing an agonist CD3-binding antibody fragment and anantagonist ICOS-binding antibody fragment, may also be employed.

Alternately, the binding pair includes monospecific antibodies, whereina first antibody binds to CD3 and a second antibody binds to CD27. Inthis embodiment, both antibodies are preferably stimulatory or agonistantibodies. It has been reported that CD27 costimulation augments thesurvival and antitumor activity of redirected human T cells in vivo(Song et al., Blood, 119(3):696-706, 2012). A bispecific antibodycontaining an agonist CD3-binding antibody fragment and an agonistCD27-binding antibody fragment, may also be employed.

Alternately, the binding pair includes monospecific antibodies, whereina first antibody binds to CD3 and a second antibody binds to CD137. Inthis embodiment, both antibodies are preferably stimulatory or agonistantibodies. It has been reported that CD137 costimulation improves theexpansion and function of CD8(+) melanoma tumor-infiltrating lymphocytesfor adoptive T-cell therapy (Chacon et al., PLoS One. 2013; 8(4):e60031,2013). A bispecific antibody containing an agonist CD3-binding antibodyfragment and an agonist CD27-binding antibody fragment, may also beemployed.

T-Cell Homeostatic Agents

In one embodiment, the MSR-SLB scaffolds and/or the antigen-presentingcell mimetic scaffolds contains a homeostatic agent is selected from thegroup consisting of IL-1, IL-2, IL-4, IL-5, IL-7, IL-10, IL-12, IL-15,IL-17, IL-21, and transforming growth factor beta (TGF-β), or an agonistthereof, a mimetic thereof, a variant thereof, a functional fragmentthereof, or a combination thereof. In some embodiments, the MSR-SLBscaffolds and/or the antigen-presenting cell mimetic scaffolds containsa plurality of homeostatic agents selected from the group consisting ofIL-1, IL-2, IL-4, IL-5, IL-7, IL-10, IL-12, IL-15, IL-17, IL-21, andtransforming growth factor beta (TGF-β), or an agonist thereof, amimetic thereof, a variant thereof, a functional fragment thereof, or acombination thereof. Functional fragments of these homeostatic agents,which are characterized by their ability to modulate the activity oftarget cells, may also be employed. Representative types of homeostaticagents, including, NCBI accession numbers of human and/or mouse homologsthereof, are provided in Table 1.

TABLE 1 Types of T-cell homeostatic agents that may be employed in thescaffolds. T-cell homeostats NCBI Accession Nos. IL-1 (IL-1α)NP_000566.3 (human) NP_034684.2 (mouse) IL-1 (IL-1β) NP_000567.1 (human)NP_032387.1 (mouse) IL-2 NP_000577.2 (human) NP_032392.1 (mouse) IL-4NP_000580.1; NP_758858.1 (human) NP_067258.1 (mouse) IL-5 NP_000870.1(human) NP_034688.1 (mouse) IL-7 NP_000871.1; NP_001186815.1;NP_001186816.1; NP_001186817.1 (human) NP_032397.1 (mouse) IL 10NP_000563.1 (human) NP_034678.1 (mouse) IL 12A NP_000873.2 (human)NP_001152896.1; NP_032377.1 (mouse) IL-12B NP_002178.2 (human)NP_001290173.1 (mouse) IL-15 NP_000576.1; NP_751915.1 (human)NP_001241676.1; NP_032383.1 (mouse) IL-17 (A) NP_002181.1; NP_034682.1(human) NP_002181.1; NP_034682.1 (mouse) TGF-beta 1 NP_000651.3 (human)NP_035707.1 (mouse) TGF-beta 2 NP_001129071.1; NP_003229.1 (human)NP_033393.2 (mouse) TGF-beta 3 NP_003230.1 (human)

Fragments and variants of the aforementioned T-cell homeostatic agentsare known in the art. For example, the UNIPROT database entry of each ofthe aforementioned homeostatic agents lists “natural variants,”including structural relationship between the variant and the wild-typebiomarker. Purely as representation, the human IL-Iβ protein (UNIPROT:P01584) includes a natural variant (VAR_073951) having E→N amino acidsubstitution at amino acid residue 141 of the putative humanIL-Iβprotein sequence. Fragments, if known, are similarly listed underthis section.

Preferably, the T-cell homeostatic agent is interleukin-2 (IL-2) or anagonist thereof, a mimetic thereof, a variant thereof, a functionalfragment thereof, or a combination thereof with one or more T-cellhomeostatic agents listed in Table 1. Examples of IL-2 agonists include,for example, BAY 50-4798 (Margolin et al., Clin Cancer Res. 2007 Jun. 1;13(11):3312-9). Examples of IL-2 mimetics include, for example, peptide1-30 (P1-30), which acts in synergy with IL-2 (Eckenberg et al., JImmunol 2000; 165:4312-4318). Examples of IL-2 fragments include, forexample, a ballast portion containing the first 100 amino acids of IL-2(see, U.S. Pat. No. 5,496,924). Examples of IL-2 variants include, forexample, natural variant VAR_003967 and natural variant VAR_003968. Alsoincluded are fusion proteins containing IL-2, e.g., F16-IL2, which is anscFv against the extra-domain A1 of tenascin-C that is fused, via ashort 5-amino acid linker, to a recombinant form of the human IL-2. Themonoclonal antibody portion of the F16-IL2 fusion protein binds to tumorcells expressing the tumor associated antigen (TAA) tenascin-C. In turn,the IL-2 moiety of the fusion protein stimulates natural killer (NK)cells, macrophages and neutrophils and induces T-cell antitumor cellularimmune responses. Other IL-2 mimetics that may be employed in accordancewith the invention include, for example, an IL-2 superkine peptide(Levin et al., Nature 484, 529-533, 2012), and an IL-2 partial agonistpeptide (Zurawski et al., EMBO Journal, 9(12): 3899-3905, 1990 and U.S.Pat. No. 6,955,807), or a combination thereof.

Embodiments of the instant invention further include MSR-SLB scaffolds,including, APC-MS scaffolds made from such scaffolds, which furthercomprise a plurality of the aforementioned T-cell homeostatic agents.Thus, in one embodiment, the invention provides for MSR-SLB scaffoldscontaining a first T-cell homeostatic agent which is IL-2 and a secondT-cell homeostatic agent which is IL-7, IL-21, IL-15, or IL-15superagonist. In this context, IL-15 superagonist (IL-15 SA) is acombination of IL-15 with soluble IL-15 receptor-α, which possessesgreater biological activity than IL-15 alone. IL-15 SA is considered anattractive antitumor and antiviral agent because of its ability toselectively expand NK and memory CD8+ T (mCD8+ T) lymphocytes. See, Guoet al., J Immunol. 2015 Sep. 1; 195(5):2353-64.

Embodiments of the instant invention further relate to scaffolds whichcomprise a plurality of T-cell stimulatory molecules, T-cellco-stimulatory molecules and T-cell homeostatic agents. A typicalscaffold may comprise at least 2, at least 3, at least 4, at least 5, atleast 6, at least 7, at least 8, at least 9, at least 10, at least 11,or more of each of the aforementioned T-cell stimulatory molecules,T-cell co-stimulatory molecules and T-cell homeostatic agents.

In the scaffolds of the invention, any functional molecule, for example,antigens, antibodies, proteins, enzymes, including fragments thereof,may be directly or indirectly immobilized onto the MSR base layer and/orthe SLB using routine techniques. In certain embodiments, the functionalmolecules may be provided in an organelle (e.g., golgi membrane orplasma membrane), a cell, a cell cluster, a tissue, a microorganism, ananimal, a plant, or an extract thereof, which in turn is immobilizedonto the MSR layer or the SLB layer. A functional molecule may also besynthesized by genetic engineering or chemical reactions at the desiredsitus, e.g., outer face of the SLB layer.

The scaffolds described herein comprise and release signaling molecules,e.g., T-cell homeostatic agents, to elicit functional T-cell responses.In one embodiment, the released T-cell homeostatic agents arepolypeptides that are isolated from endogenous sources or synthesized invivo or in vitro. For instance, endogenous IL-2 polypeptides may beisolated from healthy human tissue. Alternately, synthetic functionalmolecules may be synthesized via transfection or transformation oftemplate DNA into a host organism or cell, e.g., a cultured human cellline or a mammal (e.g., humanized mouse or rabbit). Alternatively,synthetic functional molecules in protein form may be synthesized invitro by polymerase chain reaction (PCR) or other art-recognized methodsSambrook, J., Fritsch, E. F., and Maniatis, T., Molecular Cloning: ALaboratory Manual. Cold Spring Harbor Laboratory Press, NY, Vol. 1, 2, 3(1989), incorporated by reference herein).

The functional molecules may be modified to increase protein stabilityin vivo. Alternatively, the functional molecules are engineered to bemore or less immunogenic. For instance, insofar as the structures of thevarious functional molecules are known, the sequences may be modified atone or more of amino acid residues, e.g., glycosylation sites, togenerate immunogenic variants.

In one embodiment, the functional molecules are recombinant.Alternatively, the functional molecules are humanized derivatives ofmammalian counterparts. Exemplary mammalian species from which thefunctional molecules are derived include, but are not limited to, mouse,rat, hamster, guinea pig, ferret, cat, dog, monkey, or primate. In apreferred embodiment, the functional molecules are human or humanizedversion of the aforementioned functional molecules.

Each of the aforementioned functional molecules, e.g., T-cellstimulatory molecules, T-cell co-stimulatory molecules and T-cellhomeostatic agents, may, independently from one another, be adsorbed orintegrated into the MSR base layer or the SLB base layer. Therefore, inone embodiment, there is provided an APC-MS, wherein the T-cellstimulatory molecules are adsorbed or integrated into the MSR baselayer. Preferably, there is provided an APC-MS, wherein the T-cellstimulatory molecules are adsorbed or integrated into the SLB layer. Inanother embodiment, there is provided an APC-MS, wherein the T-cellstimulatory molecules are adsorbed or integrated into both the MSR baselayer as well as the SLB layer. In another embodiment, there is providedan APC-MS, wherein the T-cell co-stimulatory molecules are adsorbed orintegrated into the MSR base layer. Preferably, there is provided anAPC-MS, wherein the T-cell co-stimulatory molecules are adsorbed orintegrated into the SLB layer. Yet in another embodiment, there isprovided an APC-MS, wherein the T-cell co-stimulatory molecules areadsorbed or integrated into both the MSR base layer as well as the SLBlayer. In another embodiment, there is provided an APC-MS, wherein theT-cell homeostatic agents are adsorbed or integrated into the MSR baselayer. In another embodiment, there is provided an APC-MS, wherein theT-cell homeostatic agents are adsorbed or integrated into the SLB layer.Yet in another embodiment, there is provided an APC-MS, wherein theT-cell homeostatic agents are adsorbed or integrated into both the MSRbase layer as well as the SLB layer.

In general, the functional molecules and the MSR base layer and/or theSLB layer, may be linked together through the use of reactive groups,which are typically transformed by the linking process into a neworganic functional group or unreactive species. The reactive functionalgroup(s), may be located in any of the aforementioned components.Reactive groups and classes of reactions useful in practicing thepresent invention are generally those that are well known in the art ofbioconjugate chemistry. Currently favored classes of reactions availablewith reactive chelates are those that proceed under relatively mildconditions. These include, but are not limited to nucleophilicsubstitutions (e.g., reactions of amines and alcohols with acyl halides,active esters), electrophilic substitutions (e.g., enamine reactions)and additions to carbon-carbon and carbon-heteroatom multiple bonds(e.g., Michael reaction, Diels-Alder addition). These and other usefulreactions are discussed in, for example, March, Advanced OrganicChemistry, 3rd Ed., John Wiley & Sons, New York, 1985; Hermanson,Bioconjugate Techniques, Academic Press, San Diego, 1996; and Feeney etal., Modification of Proteins; vol. 198, American Chemical Society,Washington, D.C., 1982.

Useful reactive pendant functional groups include, for example:

-   -   (a) carboxyl groups and various derivatives thereof including,        but not limited to, N-hydroxysuccinimide esters,        N-hydroxybenztriazole esters, acid halides (e.g., I, Br, Cl),        acyl imidazoles, thioesters, p-nitrophenyl esters, alkyl,        alkenyl, alkynyl and aromatic esters;    -   (b) hydroxyl groups, which can be converted to, e.g., esters,        ethers, aldehydes, etc.    -   (c) haloalkyl groups, wherein the halide can be later displaced        with a nucleophilic group such as, for example, an amine, a        carboxylate anion, thiol anion, carbanion, or an alkoxide ion,        thereby resulting in the covalent attachment of a new group at        the functional group of the halogen atom;    -   (d) dienophile groups, which are capable of participating in        Diels-Alder reactions such as, for example, maleimido groups;    -   (e) aldehyde or ketone groups, such that subsequent        derivatization is possible via formation of carbonyl derivatives        such as, for example, imines, hydrazones, semicarbazones or        oximes, or via such mechanisms as Grignard addition or        alkyllithium addition;    -   (f) sulfonyl halide groups for subsequent reaction with amines,        for example, to form sulfonamides;    -   (g) thiol groups, which can be, for example, converted to        disulfides or reacted with acyl halides;    -   (h) amine or sulfhydryl groups, which can be, for example,        acylated, alkylated or oxidized;    -   (i) alkenes, which can undergo, for example, cycloadditions,        acylation, Michael addition, etc;    -   (j) epoxides, which can react with, for example, amines and        hydroxyl compounds; and    -   (k) phosphoramidites and other standard functional groups useful        in nucleic acid synthesis.

The reactive functional groups can be chosen such that they do notparticipate in, or interfere with, the reactions necessary to assemblethe reactive chelates. Alternatively, a reactive functional group can beprotected from participating in the reaction by the presence of aprotecting group. Those of skill in the art understand how to protect aparticular functional group such that it does not interfere with achosen set of reaction conditions. See, for example, Greene et al.,Protective Groups in Organic Synthesis, John Wiley & Sons, New York,1991.

In one embodiment, the functional molecules are loaded/adsorbed onto theMSR base layer or the SLB or both the MSR layer and the SLB via affinitypairing or chemical coupling.

The term “affinity pair” as used herein includes antigen-antibody,receptor-hormone, receptor-ligand, agonist-antagonist,lectin-carbohydrate, nucleic acid (RNA or DNA) hybridizing sequences, Fcreceptor or mouse IgG-protein A, avidin-biotin, streptavidin-biotin,biotin/biotin binding agent, Ni2+ or Cu2+/HisTag (6× histidine) andvirus-receptor interactions. Various other specific binding pairs arecontemplated for use in practicing the methods of this invention.

As used herein, “biotin binding agent” encompasses avidin, streptavidinand other avidin analogs such as streptavidin or avidin conjugates,highly purified and fractionated species of avidin or streptavidin, andnon or partial amino acid variants, recombinant or chemicallysynthesized avidin analogs with amino acid or chemical substitutionswhich still accommodate biotin binding. Preferably, each biotin bindingagent molecule binds at least two biotin moieties and more preferably atleast four biotin moieties. As used herein, “biotin” encompasses biotinin addition to biocytin and other biotin analogs such as biotin amidocaproate N-hydroxysuccinimide ester, biotin 4-amidobenzoic acid,biotinamide caproyl hydrazide and other biotin derivatives andconjugates. Other derivatives include biotin-dextran,biotin-disulfide-N-hydroxysuccinimide ester, biotin-6 amido quinoline,biotin hydrazide, d-biotin-N hydroxysuccinimide ester, biotin maleimide,d-biotin p-nitrophenyl ester, biotinylated nucleotides and biotinylatedamino acids such as Nε-biotinyl-1-lysine.

The ligands that may be functionalized via affinity pairing include, butare not limited to, receptors, monoclonal or polyclonal antibodies,viruses, chemotherapeutic agents, receptor agonists and antagonists,antibody fragments, lectin, albumin, peptides, proteins, hormones, aminosugars, lipids, fatty acids, nucleic acids and cells prepared orisolated from natural or synthetic sources. In short, any site-specificligand for any molecular epitope or receptor to be detected through thepractice of the invention may be utilized. Preferably, the ligand is amembrane-anchored protein. The ligand may also be a derivative of amembrane-anchored protein, such as a soluble extracellular domain. Aligand can be a receptor involved in receptor-receptor cellularinteractions such as TCR binding to the MHC receptor.

The ligands of the instant invention can be expressed and purified byany method known in the art. In a certain embodiment, the proteins areexpressed by a baculovirus-based insect expression system or a mammalianexpression system. Fifteen residues of AVITAG™ peptide may be added tothe C-terminals of all of the molecules. The lysine residue in theAVITAG™ (Avidity, CO) can be specifically biotinylated by BirA enzyme(Avidity, CO). The proteins may also be designed to be secreted into thesupernatant of the cell culture.

The functional molecules, as noted hereinabove, can be any protein orpeptide. Preferably, the proteins are involved in ligand-receptorinteractions. For example, an important event of T cell activation is aresult of membrane-membrane contact between T cells and APCs, wherein avariety of ligand-receptor interactions take place between the twoopposing membranes, including, MHC-peptide and TCR, LFA-1 and ICAM-1,CD2 and CD48, as well as B7 or CTLA-4 and CD28. Understanding thevalency requirements of these interactions will facilitate the design oftherapeutics that enhance or inhibit the immune response to certainantigens. The instant invention can also be used as a tool to study thesubtle differences in T cell intracellular signaling pathways induced byagonist and antagonist antigens. The scaffolds provide a cleanphysiological setting to test the subtle differences without usingnative antigen presenting cells that often complicate biochemicalanalyses.

While streptavidin-biotin interactions are exemplified throughout thespecification and examples, specific binding pair members as describedhereinabove may be employed in place of streptavidin and biotin in themethods of the instant invention. Furthermore, more than one set ofspecific binding pairs can be employed, particularly when more than oneligand is attached to the membrane surface. In this context, traditionalpep-MHC-streptavidin tetramer technology can also be used to screen Tcells of certain pep-MHC specificity. However, T cells with the samespecificity may or may not be activated by the same antigen stimulation.To study immune responses (e.g. responses to vaccination [viral orcancer vaccines], immune tolerance, autoimmunity), it is important todiscriminate T cells based on their responsiveness to antigen. Usingcalcium flux by microscopy as an indicator for T cell activation, theinstant invention also provides a screening assay to quantify primary Tcells responsive to a specific antigen. Alternately, biotinylatedpep-MHC and co-stimulatory molecules may be coupled onto a streptavidincoated chips, and the chips are paired with the scaffolds of theinvention.

In another embodiment, the functional molecules are chemically coupledto the MSR base layer and/or the SLB layer. In certain embodiments, thechemical coupling includes, click-chemistry reagents, for example,azide-alkyne chemical (AAC) reaction, dibenzo-cyclooctyne ligation(DCL), or tetrazine-alkene ligation (TAL). For instance, in the contextof AAC, either the MSR or the SLB contains a plurality of single clickchemistry functionalities, and frequently contains two, three or more ofsuch functionalities. One or two such functionalities per molecule arepreferred. In one embodiment, a clickable reagent such as3-azidopropylamine or 10-undecynoic acid may be amide-bonded to thecarboxy- or amino-terminus, respectively, of a peptide or protein via aclick reaction with a corresponding alkyne or azido compound andappropriate catalyst to form the 1,2,3-triazole ring linking groups.See, e.g., U.S. Publication No. 2007/0060658. To further extend arsenalof bioorthogonal copper-free click reagents, aza-dibenzocyclooctyne(ADIBO)-containing compounds for azide-coupling reactions may be usedfor the site-specific covalent anchoring of protein functionalmolecules, e.g., antibodies, interluekins and cytokines. The samemetal-free click reaction is employed for the PEGylation ofunfunctionalized areas of the surface. Such treatment allows for adramatic reduction or complete elimination of non-specific binding. Thecopper-free click immobilization methods can be applied to thepreparation of various types of arrays, as well as to the derivatizationof microbeads and nanoparticles. See, e.g., U.S. Pat. No. 8,912,322. Insome embodiments, the functional molecules are coupled to the MSR baselayer and/or the SLB layer using a click reagent selected from the groupconsisting of azide, dibenzocyclooctyne (DBCO), transcyclooctene,tetrazine and norbornene and variants thereof. In some embodiments, thefunctional molecule comprises azide and a lipid of the lipid bilayer ofthe MSR-SLB comprises DBCO.

The term “click chemistry” refers to a chemical philosophy introduced byK. Barry Sharpless of The Scripps Research Institute, describingchemistry tailored to generate covalent bonds quickly and reliably byjoining small units comprising reactive groups together. Click chemistrydoes not refer to a specific reaction, but to a concept includingreactions that mimic reactions found in nature. In some embodiments,click chemistry reactions are modular, wide in scope, give high chemicalyields, generate inoffensive byproducts, are stereospecific, exhibit alarge thermodynamic driving force >84 kJ/mol to favor a reaction with asingle reaction product, and/or can be carried out under physiologicalconditions. A distinct exothermic reaction makes a reactant “springloaded”. In some embodiments, a click chemistry reaction exhibits highatom economy, can be carried out under simple reaction conditions, usereadily available starting materials and reagents, uses no toxicsolvents or use a solvent that is benign or easily removed (preferablywater), and/or provides simple product isolation by non-chromatographicmethods (crystallization or distillation).

The term “click chemistry handle,” as used herein, refers to a reactant,or a reactive group, that can partake in a click chemistry reaction. Forexample, a strained alkyne, e.g., a cyclooctyne, is a click chemistryhandle, since it can partake in a strain-promoted cycloaddition. Ingeneral, click chemistry reactions require at least two moleculescomprising click chemistry handles that can react with each other. Suchclick chemistry handle pairs that are reactive with each other aresometimes referred to herein as partner click chemistry handles. Forexample, an azide is a partner click chemistry handle to a cyclooctyneor any other alkyne. Exemplary click chemistry handles suitable for useaccording to some aspects of this invention are described herein, forexample, US 2014/0249296. Other suitable click chemistry handles areknown to those of skill in the art.

In one embodiment, the instant invention provides APC-MS comprising aplurality of T-cell activating molecules and T-cell co-stimulatorymolecules optionally together with T-cell homeostatic agents, which areadsorbed into the scaffold via metal-chelating lipid headgroups. See,Maloney et al., Chem Biol., 3(3):185-92, 1996. Several approaches usingchelated metal ions have been reported that allow histidine-taggedproteins to be immobilized at several types of interfaces, such as lipidinterfaces and lipid monolayers with metal-chelating lipids, goldsurfaces with self-assembling monolayers formed with metal-chelatingalkanethiols, and oxide surfaces with metal-chelating silanes. Forexample, Peterson et al. (U.S. Pat. No. 5,674,677) describes a methodfor joining two amino acid sequences by coupling an organic chelator toa protein, e.g., an enzyme, and charging the chelator with a metal ion.This complex is then mixed with any protein containing a histidine tagto couple the complex with the histidine tagged protein. See also, U.S.Pat. No. 6,087,452, which is incorporated by reference herein in itsentirety.

The functional molecules of the invention are preferably proteins. Theterms “protein,” “peptide” and “polypeptide” are used interchangeably,and refer to a polymer of amino acid residues linked together by peptide(amide) bonds. The terms refer to a protein, peptide, or polypeptide ofany size, structure, or function. Typically, a protein, peptide, orpolypeptide will be at least three amino acids long. A protein, peptide,or polypeptide may refer to an individual protein or a collection ofproteins. One or more of the amino acids in a protein, peptide, orpolypeptide may be modified, for example, by the addition of a chemicalentity such as a carbohydrate group, a hydroxyl group, a phosphategroup, a farnesyl group, an isofarnesyl group, a fatty acid group, alinker for conjugation, functionalization, or other modification, etc. Aprotein, peptide, or polypeptide may also be a single molecule or may bea multi-molecular complex. A protein, peptide, or polypeptide may bejust a fragment of a naturally occurring protein or peptide. A protein,peptide, or polypeptide may be naturally occurring, recombinant, orsynthetic, or any combination thereof.

The term “conjugated” or “conjugation” refers to an association of twomolecules, for example, two proteins, with one another in a way thatthey are linked by a direct or indirect covalent or non-covalentinteraction. In the context of conjugation via click chemistry, theconjugation is via a covalent bond formed by the reaction of the clickchemistry handles. In certain embodiments, the association is covalent,and the entities are said to be “conjugated” to one another. In someembodiments, a protein is post-translationally conjugated to anothermolecule, for example, a second protein, by forming a covalent bondbetween the protein and the other molecule after the protein has beentranslated, and, in some embodiments, after the protein has beenisolated. In some embodiments, the post-translational conjugation of theprotein and the second molecule, for example, the second protein, iseffected via installing a click chemistry handle on the protein, and asecond click chemistry handle, which can react to the first clickchemistry handle, on the second molecule, and carrying out a clickchemistry reaction in which the click chemistry handles react and form acovalent bond between the protein and the second molecule, thusgenerating a chimeric protein. In some embodiments, two proteins areconjugated at their respective C-termini, generating a C-C conjugatedchimeric protein. In some embodiments, two proteins are conjugated attheir respective N-termini, generating an N-N conjugated chimericprotein.

In certain embodiments, a plurality of detectable labels may be used toanalyze and/or study the conjugation process. As used herein, a“detectable label” refers to a moiety that has at least one element,isotope, or functional group incorporated into the moiety which enablesdetection of the molecule, e.g., a protein or polypeptide, or otherentity, to which the label is attached. Labels can be directly attached(i.e., via a bond) or can be attached by a tether (such as, for example,an optionally substituted alkylene; an optionally substitutedalkenylene; an optionally substituted alkynylene; an optionallysubstituted heteroalkylene; an optionally substituted heteroalkenylene;an optionally substituted heteroalkynylene; an optionally substitutedarylene; an optionally substituted heteroarylene; or an optionallysubstituted acylene, or any combination thereof, which can make up atether). It will be appreciated that the label may be attached to orincorporated into a molecule, for example, a protein, polypeptide, orother entity, at any position.

In general, a label can fall into any one (or more) of five classes: a)a label which contains isotopic moieties, which may be radioactive orheavy isotopes, including, but not limited to, ²H, ³H, ¹³C, ¹⁴C, ¹⁵N,¹⁸F, ³¹P, ³²P, ³⁵S, ⁶⁷Ga, ⁹⁹mTc (Tc-99 m), ¹¹¹In, ¹²⁵I, ¹³¹I, ¹⁵³Gd,¹⁶⁹Yb, and ¹⁸⁶Re; b) a label which contains an immune moiety, which maybe antibodies or antigens, which may be bound to enzymes (e.g., such ashorseradish peroxidase); c) a label which is a colored, luminescent,phosphorescent, or fluorescent moieties (e.g., such as the fluorescentlabel fluorescein isothiocyanate (FITC) or carboxyfluorescein); d) alabel which has one or more photo affinity moieties; and e) a labelwhich is a ligand for one or more known binding partners (e.g.,biotin-streptavidin, FK506-FKBP). In certain embodiments, a labelcomprises a radioactive isotope, preferably an isotope which emitsdetectable particles. In certain embodiments, the label comprises afluorescent moiety. In certain embodiments, the label is the fluorescentlabel fluorescein isothiocyanate (FITC). In certain embodiments, thelabel comprises a ligand moiety with one or more known binding partners.In certain embodiments, the label comprises biotin. In some embodiments,a label is a fluorescent polypeptide (e.g., GFP or a derivative thereofsuch as enhanced GFP (EGFP)) or a luciferase (e.g., a firefly, Renilla,or Gaussia luciferase). It will be appreciated that, in certainembodiments, a label may react with a suitable substrate (e.g., aluciferin) to generate a detectable signal. Non-limiting examples offluorescent proteins include GFP and derivatives thereof, proteinscomprising chromophores that emit light of different colors such as red,yellow, and cyan fluorescent proteins, etc. Exemplary fluorescentproteins include, e.g., Sirius, Azurite, EBFP2, TagBFP, mTurquoise,ECFP, Cerulean, TagCFP, mTFP1, mUkG1, mAG1, AcGFP1, TagGFP2, EGFP,mWasabi, EmGFP, TagYPF, EYFP, Topaz, SYFP2, Venus, Citrine, mKO, mKO2,mOrange, mOrange2, TagRFP, TagRFP-T, mStrawberry, mRuby, mCherry,mRaspberry, mKate2, mPlum, mNeptune, T-Sapphire, mAmetrine, mKeima. See,e.g., Chalfie, M. and Kain, S R (eds.) Green fluorescent protein:properties, applications, and protocols (Methods of BiochemicalAnalysis, v. 47). Wiley-Interscience, Hoboken, N.J., 2006, and/orChudakov et al., Physiol Rev. 90(3):1103-63, 2010 for discussion of GFPand numerous other fluorescent or luminescent proteins. In someembodiments, a label comprises a dark quencher, e.g., a substance thatabsorbs excitation energy from a fluorophore and dissipates the energyas heat.

In another embodiment, the functional molecules may be loaded ontomesoporous silica and/or the lipid bilayer using art known, covalent ornon-covalent loading techniques. In one embodiment, the functionalmolecules are loaded non-covalently. For instance, Lei et al. (U.S.Publication No. 2011-0256184) describe mesoporous silicates that provideenhanced, spontaneous loading of antibodies such as IgG via non-covalentbonding within the native or functionalized structure. Accordingly, thescaffolds of the invention may be formulated with such silicates.

In another embodiment, the functional molecules are chemically coupledonto the MSR. In such embodiments, the coupling may be conducted byutilizing one or more of the following molecules and the reactive groupscontained therein: cysteine (thiol group), serine or threonine (hydroxylgroup), lysine (amino group), aspartate or glutamate (carboxyl group).Alternatively, the functional molecules may be conjugated to the MSR viautilization of polyhistidine-tag (His-tag), a peptide containingpolyhistidine-tag or an antibody containing polyhistidine-tag. Herein,the polyhistidine-tag consists of at least four, five, six or sevenhistidine (His) residues.

In one embodiment, an anchor is used to connect the functional moleculeto a pore wall. However, the anchor is not an essential component. Incertain embodiments, each pore of the mesoporous silica accommodates atleast one functional molecule. Thus, the pores must have a sizeappropriate to immobilize a biological substance. The pore size dependson the size of the functional molecule to be immobilized. When afunctional molecule is immobilized in a pore, the functional moleculecan be adsorbed on an inner surface of the pore by electrostaticbonding. A functional molecule may also be held in a pore by anoncovalent bonding, such as van der Waals forces, hydrogen bonding, orionic bonding.

In the aforementioned embodiment where the MSR comprises anchoringmoieties, the anchor may have an effect of reducing a large structuralchange of the functional molecule to hold it stably. Preferably, theanchor is composed of substantially the same component as the mesoporousmaterial. The anchor may comprise one or more functional groups topermit binding to a desired functional molecule: a hydroxyl group, anamide group, an amino group, a pyridine group, a urea group, a urethanegroup, a carboxyl group, a phenol group, an azo group, a hydroxyl group,a maleimide group, a silane derivative, or an aminoalkylene group.

Embodiments of the invention further relate to MSR-SLB scaffolds of theinvention, including, scaffolds containing such scaffolds, comprising, aplurality of the aforementioned functional molecules which are adsorbedin the lipid matrix.

In one embodiment, the functional molecules are adsorbed into thesupported lipid bilayer via physical insertion. Techniques for insertingproteins into the bilayer of amphipathic molecules are known in the art.In one embodiment, proteins in the environment of the bilayer, forexample in the hydrophobic medium and/or in the hydrophilic body and/orin the hydrated support, may insert spontaneously into the bilayer.Alternatively, proteins may be driven into the bilayer by theapplication of a voltage and/or by fusion of protein loaded vesicleswith the bilayer. The vesicles may be contained within or introduced tothe hydrophilic body. In one instance, proteins may be introduced intothe membrane by using the probe method disclosed in PCT Publication No.

WO 2009/024775. The inserted protein may be a known membrane-associatedprotein, e.g., one or more of the aforementioned T-cell activatingmolecules and/or T-cell co-stimulatory molecules.

In another embodiment, the functional molecule may be an antigen that isused in expansion of T-cells. Representative examples of such antigensusable in T-cell expansion include, full-length CD19 or a fragmentthereof or a variant thereof. CD19 is a prototypical antigen used in theexpansion of chimeric antigen receptor (CAR) T-cells. See, Turtle etal., Blood, 126:184, 2015; Turtle et al., J Clin Invest., 126, 2123-38,2016. In another embodiment, the antigen is full-length CD22 or afragment thereof or a variant thereof, which are also useful in theexpansion of CAR T-cells. See, Haso et al., Blood, 121(7): 1165-1174,2013; Qin et al., Blood, 122:1431, 2013.

In an alternate embodiment, the functional molecule may be amembrane-associated protein which is anchored directly or indirectly tothe bilayer. Other functional molecules, e.g., selective ornon-selective membrane transport proteins, ion channels, pore formingproteins or membrane-resident receptors, etc. may also be inserted intothe SLB via this method.

In another embodiment, the functional molecules may be conjugated tomembrane-associated proteins which associate with and/or insert into theSLB, e.g. gramicidin; α-helix bundles, e.g. bacteriorhodopsin or K+channels; and β-barrels, e.g., α-hemolysin, leukocidin or E. coliporins; or combinations thereof.

In certain embodiments, the fabricated SLB (containing one or morefunctional molecules) may be stabilized by compounds such as ionic ornon-ionic surfactants. Suitable surfactants include, but are not limitedto, the following examples: synthetic phospholipids, their hydrogenatedderivatives and mixtures thereof, sphingolipids and glycosphingolipids,saturated or unsaturated fatty acids, fatty alcohols,polyoxyethylene-polyoxypropylene copolymers, ethoxylated fatty acids aswell as esters or ethers thereof, dimyristoyl phosphatidyl choline,dimyristoyl phosphatidyl glycerol or a combination of two or more of theabove mentioned. A preferred surfactant according to the invention isthe dimyristoyl phosphatidyl glycerol.

The fabricated SLBs may be optionally stabilized by at least oneco-surfactant selected in the group comprising or consisting of butanol,butyric acid, hexanoic acid, sodium cholate, sodium taurocholate andsodium glycocholate, more particularly sodium cholate.

The fabricated SLBs may also include other excipients, such as polymershaving bioadhesive or absorption enhancing properties and selected fromthe group comprising or consisting of acrylic polymers (CARBOPOL®,Polycarbophil, NOVEON®), medium chain fatty acids and polyethyleneglycols. Preferred excipients are the above-mentioned acrylic polymers.

The SLB may be modified with reagents for detecting membrane-associatedproteins. Preferably the membrane-associated proteins are ion channelproteins and/or pore forming proteins. Preferably themembrane-associated proteins diffuse into and/or associate with thebilayer causing a detectable change in the properties at the bilayer.The properties changed may be physical, optical, electrical orbiochemical.

In some embodiments, the MSR-SLB scaffolds and/or the antigen-presentingcell mimetic scaffolds comprises a small molecule drug. In someembodiments, the MSR-SLB scaffolds and/or the antigen-presenting cellmimetic scaffolds comprises a thalomid analog. In some embodiments, theMSR-SLB scaffolds and/or the antigen-presenting cell mimetic scaffoldscomprises a IDO/MEK inhibitor. In some embodiments, the MSR-SLBscaffolds and/or the antigen-presenting cell mimetic scaffolds comprisesa small molecule drug that has immunomodulatory effects. Small moleculedrugs with immunomodulatory effects are known the art (see, e.g., Murphyet al. Hum. Vaccin. Immunother. 11(10): 2463-8 (2015), the entirecontents of which are expressly incorporated herein by reference).

In certain embodiments, the MSR-SLB scaffolds containing the functionalmolecules may be used to detect cells which are capable of interactionwith amphipathic molecules in the bilayer and/or with the functionalmolecule in the bilayer. The interaction may be specific or non-specificin nature. Alternatively the cells may interact with the functionalmolecule or with the lipid bilayer to cause physical, optical,electrical, or biochemical changes. Such interaction may be detected inmany different ways, including, but limited to, by visual changes, viaactivation of fluorescently labelled lipids or proteins in the SLB, orchanges in capacitance of the SLB.

Biodegradable Scaffolds

Embodiments of the invention further relate to biodegradable scaffolds.In one embodiment, the scaffold structure may substantially degrade whenexposed to a biological milieu. In one embodiment, the biological milieuis a tissue culture condition, e.g., tissue culture media that has beenoptionally adapted to culture lymphocytes such as T-cells. In anotherembodiment, the biological milieu is a biological fluid, e.g., blood,lymph, CSF, peritoneal fluid, or the like. In yet another embodiment,the biological milieu is the tissue environment at the site of implant,e.g., blood vessels, lymphatic system, adipose tissue, or the like.

In certain embodiments, the biodegradable scaffolds are substantiallydegraded following contact with a biological milieu in vivo over 1 day,2 days, 3 days, 4 days, 5 days, 6 days, 7, days, 8 days, 9 days, 10days, 11 days, 12 days, 13 days, 14 days, 15 days, 20 days, 30 days, 45days, 60 days, 90 days, or more. In certain embodiments, thebiodegradable scaffolds are substantially degraded following contactwith a biological milieu in vivo in less than 1 week. In certainembodiments, the biodegradable scaffolds are substantially degradedfollowing contact with a biological milieu in vitro over 1 day, 2 days,3 days, 4 days, 5 days, 6 days, 7, days, 8 days, 9 days, 10 days, 11days, 12 days, 13 days, 14 days, 15 days, 20 days, 30 days, 45 days, 60days, 90 days, or more. In certain embodiments, the biodegradablescaffolds are substantially degraded following contact with a biologicalmilieu in vitro in less than 1 week. By substantial degradation, it ismeant that at least 30%, at least 50%, at least 60%, at least 70%, atleast 90%, at least 95%, or more of the scaffold composition is degradedwhen the scaffold composition is contacted with the biological milieu.

In certain embodiments, it may be advantageous to use biodegradablescaffolds. For instance, by fabricating the scaffold composition suchthat it substantially degrades during the incubation period (e.g., whenthe T-cells are allowed to expand), it may be possible to use theexpanded T-cells without subjecting them to additional purificationand/or formulation steps. Avoiding downstream purification and/orformulation steps would ensure that the T-cells are fit and possess thedesired functionality for the desired application.

Accordingly, in certain embodiments, it may be advantageous to tailorthe degradation kinetics of the scaffold compositions by modifying theproperties of mesoporous silica rods, such as size, geometry, porosity.Alternately, the degradation kinetics of the scaffold compositions maybe modified by changing the culture conditions (e.g., by adjusting thepH of the media).

In accordance with the aforementioned objectives, embodiments of theinvention relate to MSR-SLB scaffolds comprising a plurality offunctional molecules which are optionally biodegradable. In oneembodiment, the scaffolds of the instant invention may be encapsulatedinto other biodegradable scaffolds. Reagents and techniques that areuseful in making such composite biodegradable scaffold compositions areknown in the art. See, Liao et al., J. Biomed. Mater. Res. B. Appl.Biomater., 102(2):293-302, 2014. In one embodiment, the scaffolds aremade up of physiologically-compatible and optionally biodegradablepolymers. Examples of polymers that are employable in the scaffolds areknown in the art. See, for example, U.S. Publication No. 2011/0020216,the entire contents of which are incorporated herein by reference.Representative examples of such polymers include, but are not limitedto, poly(lactide)s, poly(glycolide)s, poly(lactic acid)s, poly(glycolicacid)s, polyanhydrides, polyorthoesters, polyetheresters,polycaprolactones, polyesteramides, polycarbonates, polycyanoacrylates,polyurethanes, polyacrylates, and blends or copolymers thereof.Biodegradable scaffolds may comprise biodegradable materials, e.g.,collagen, alginates, polysaccharides, polyethylene glycol (PEG),poly(glycolide) (PGA), poly(L-lactide) (PLA), orpoly(lactide-co-glycolide) (PLGA) or silk. Methods for fabricating thescaffold compositions are known in the art. See, for example, Martinsenet al. (Biotech. & Bioeng., 33 (1989) 79-89), (Matthew et al.(Biomaterials, 16 (1995) 265-274), Atala et al. (J Urology, 152 (1994)641-643), and Smidsrod (TIBTECH 8 (1990) 71-78), the disclosures inwhich are incorporated by reference herein.

Exemplary scaffolds utilize glycolides or alginates of a relatively lowmolecular weight, preferably of size which, after dissolution, is at therenal threshold for clearance by humans, e.g., the alginate orpolysaccharide is reduced to a molecular weight of 1000 to 80,000daltons. Preferably, the molecular mass is 1000 to 60,000 daltons,particularly preferably 1000 to 50,000 daltons. It is also useful to usean alginate material of high guluronate content since the guluronateunits, as opposed to the mannuronate units, provide sites for ioniccross-linking through divalent cations to gel the polymer. For example,U.S. Pat. No. 6,642,363, which incorporated herein by reference,discloses methods for making and using polymers containingpolysaccharides such as alginates.

The scaffolds of the invention may be porous such that the scaffolds cansustain antigen presentation and attract and manipulate immune cells. Inone embodiment, the scaffolds contain porous matrices, wherein the poreshave a diameter between 10 nm to 500 m, particularly between 100 nm and100 μm. In these embodiments, the invention utilizes scaffoldscomprising mesoporous scaffolds. Methods of making polymer matriceshaving the desired pore sizes and pore alignments are described in theart, e.g., US pub. No. 2011/0020216 and U.S. Pat. No. 6,511,650, whichare incorporated herein by reference.

The mesoporous silica rods can be modified into multifunctional deliveryplatforms for delivering drugs such as chemotherapeutic agents andDNA/siRNA, antibody and protein biologics, cells, etc. (Lee et al., Adv.Funct. Mater., 215-222, 2009; Liong et al., ACS Nano, 889-896, 2008;Meng et al., ACS Nano, 4539-4550, 2010; Meng et al., J. Am. Chem. Soc.,12690-12697, 2010; Xia et al., ACS Nano, 3273-3286, 2009; Radu et al.,J. Am. Chem. Soc., 13216-13217, 2004; Slowing et al., J. Am. Chem. Soc.,8845-8849, 2007). This delivery platform allows effective and protectivepackaging of hydrophobic and charged anticancer drugs for controlled andon demand delivery, with the additional capability to also image thedelivery site (Liong et al., ACS Nano, vol. 2, pp. 889-896, 2008). Thekey challenge now is to optimize the design features for efficient andsafe in vivo drug delivery (He et al., Small, vol. 7, pp. 271-280, 2011;Lee et al., Angew. Chem. Int. Ed., vol. 49, pp. 8214-8219, 2010; Liu etal., Biomaterials, vol. 32, pp. 1657-1668, 2011; Al Shamsi et al., Chem.Res. Toxicol., vol. 23, pp. 1796-1805, 2010), which can be assessedthrough the use of human xenograft tumors in nude mice (Lu et al.,Small, vol. 6, pp. 1794-1805, 2010).

Embodiments described herein further relate to MSR-SLB scaffolds,including, scaffolds containing such scaffolds, wherein the dry weightratio of the mesoporous silica micro-rods (MSR) to the T-cellactivating/co-stimulatory molecules is between about 1:1 to about 100:1,preferably between about 10:1 to about 50:1, particularly between about20:1 to about 50:1. In some embodiments, the dry weight ratio of themesoporous silica micro-rods (MSR) to the T-cellactivating/co-stimulatory molecules of the MSR-SLB scaffolds is betweenabout 10,000:1 to about 1:1. In some embodiments, the dry weight ratioof the mesoporous silica micro-rods (MSR) to the T-cellactivating/co-stimulatory molecules of the MSR-SLB scaffolds is betweenabout 5,000:1 to about 1:1, between about 1,000:1 to about 1:1, betweenabout 500:1 to about 1:1, between about 100:1 to about 1:1. In someembodiments, the dry weight ratio of the mesoporous silica micro-rods(MSR) to the T-cell activating/co-stimulatory molecules of the MSR-SLBscaffolds is about 10,000:1, about 5,000:1, about 2,500:1, about1,000:1, about 750:1, about 500:1, about 250:1, about 100:1, about 75:1,about 50:1, about 40:1, about 30:1, about 25:1, about 20:1, about 10:1,or about 1:1.

Embodiments described herein further relate to compositions and devicescontaining aforementioned scaffolds containing the MSR-SLB scaffoldstogether with the functional molecules, e.g., T-cell activatingmolecule, T-cell co-stimulatory molecule, and T-cell homeostatic agent,optionally together with one or more additional agents (listed below).In one embodiment, the invention provides for compositions comprisingthe scaffold and T-cells clustered therein. In one embodiment, theT-cells are selected from the group consisting of natural killer (NK)cells, a CD3+ T-cells, CD4+ T-cells, CD8+ T-cells, and regulatoryT-cells (Tregs), or a combination thereof. In other embodiments, thecomposition may be a pharmaceutical composition, which may be producedusing methods that are well-known in the art. For instance,pharmaceutical compositions may be produced by those of skill, employingaccepted principles of medicinal chemistry. The compositions, scaffolds,and devices may be provided with one or more reagents for selecting,culturing, expanding, sustaining, and/or transplanting the cells ofinterest. Representative examples of cell selection kits, culture kits,expansion kits, transplantation kits for T-cells, B-cells and antigenpresenting cells are known in the art. For example, where the targetcell of interest are T-cells, such may be initially sorted usingDYNABEADS, MACS-beads (Miltenyi Biosciences), maintained in STEMXVIVOHuman T cell base media (R&D Systems) and expanded with OPTIMIZERculture media (Thermo Fisher Scientific). The cells may be enriched inthe sample by using centrifugation techniques known to those in the artincluding, e.g., FICOLL® gradients. Cells may also be enriched in thesample by using positive selection, negative selection, or a combinationthereof, based on the expression of certain markers.

Further embodiments of the invention relate to T-cell manipulatingdevices. The devices contain the scaffolds of the invention togetherwith a plurality of molecules which attract/bind to target T cells. Inone embodiment, the invention relates to devices containing scaffoldsthat are stacked to selectively permit infiltration of T-cells into themesoporous silica micro-rods (MSR). By selective infiltration, it ismeant that owing to selective permissibility/permeability, specificityof binding, selective elimination (of undesired cells) and/or expansion(of desired cells), the scaffold contains at least 10% more, 20% more,30% more, 40% more, 50% more, 60% more, 70% more, 80% more, 90% more,100% more, 150% more, 200% more, 300% more, 400% more, 500% more, 600%more, 800% more, 1000% more, or greater number of target T-cells after aperiod of incubation compared to that which is present in whole blood.In certain embodiments, the period of incubation is between 1-30 days,preferably between 4-15 days, particularly between 7-12 days. In otherembodiments, selective infiltration relates to retention and/orexpansion of T-cells compared to other blood cells, e.g., B-cells,dendritic cells, macrophages, red blood cells or platelets that arepresent in whole blood.

In other embodiments, the scaffolds of the invention permit selectiveinfiltration of a specific sub-population of T-cells, e.g., naturalkiller (NK) cells, a CD3+ T-cells, CD4+ T-cells, CD8+ T-cells, orregulatory T-cells (Tregs). Herein, the scaffold contains at least 10%more, 20% more, 30% more, 40% more, 50% more, 60% more, 70% more, 80%more, 90% more, 100% more, 150% more, 200% more, 300% more, 400% more,500% more, 600% more, 800% more, 1000% more, or greater number of targetT-cells after 4-14 days incubation compared to that which is present inwhole blood. The percentages and the ranges of various types oflymphocytes in human whole blood are as follows: NK cells 7% (range:2-13%); helper T cells 46% (range: 28-59%); cytotoxic T cells 19%(range: 13-32%); γδ T cells 5% (range: 2%-8%); B cells 23% (range:18-47%) (Berrington et al., Clin Exp Immunol 140 (2): 289-292, 2005).

Additional Agents

The scaffolds of the invention include one or more agents, which may benaturally-occurring, synthetically produced, or recombinant compounds,e.g., peptides, polypeptides, proteins, nucleic acids, small molecules,haptens, carbohydrates, or other agents, including fragments thereof orcombinations thereof. In one embodiment, the agents are antigens. In oneembodiment, the antigens are peptides or proteins or immunologicallyactive fragments thereof. In one embodiment, the antigens describedherein are purified. Purified compounds contain at least 60% by weight(dry weight) of the compound of interest. Particularly, the antigens areat least 75% pure, preferably at least 90% pure, and more preferably atleast 99% pure. Purity is measured by any appropriate standard method,for example, by column chromatography, gel electrophoresis, or HPLCanalysis. The antigens may be self-antigens or non-self antigens.

Representative examples of non-self antigens include, for example,antigens derived from a pathogen selected from the group consisting of avirus, a bacterium, a protozoan, a parasite, and a fungus. The antigensmay be optionally loaded onto MHC molecules, e.g., HLA-A, HLA-B, HLA-C,DP, DQ and DR, which are then incorporated into the scaffolds.

Alternately, the scaffolds contain a plurality of self-antigens, whichare optionally linked to or associated with a disease or disorder.Preferably, the self-antigens are specifically associated with a humandisease or a disorder. In one embodiment, the self-antigen is associatedwith an autoimmune disorder selected from the group consisting ofrheumatoid arthritis, lupus, celiac disease, inflammatory bowel diseaseor Crohn's disease, sjögren's syndrome polymyalgia rheumatic, multiplesclerosis, ankylosing spondylitis, Type 1 diabetes, alopecia areata,vasculitis, temporal arteritis, etc. Specific types of antigens,including fragments thereof, which are associated with type 1 diabetes,multiple sclerosis, Crohn's disease, and rheumatoid arthritis and thelike have been characterized in literature. For example, rheumatoidarthritis-related antigen is a 47 kDa protein (RA-A47). See Hattori etal, J Bone Miner Metab., 18(6):328-34 (2000). In Crohn's disease, theantigen may be bacterial flagellin. See, Lodes et al., J Clin Invest.113(9):1296-306 (2004). Likewise, major myelin proteins such as myelinbasic protein (MBP) and proteolipid protein (PLP), are likely to be ofimportance in the course of multiple sclerosis (MS). See, deRosbo etal., J Clin Invest. 92(6): 2602-260 (1993). In the context of type 1diabetes, a plurality of autoantigens may be involved, such as,preproinsulin (PPI), islet-specific glucose-6-phosphatase (IGRP),glutamate decarboxylase (GAD65), insulinoma antigen-2 (IA-2),chromogranin A and heat shock protein 60. See Roep et al., Cold SpringHarb Perspect Med. 2(4), 2012 (PMID: 22474615).

In another embodiment, the self-antigens are associated with a cancer.Representative types of cancer antigens include, for example, MAGE-1,MAGE-2, MAGE-3, CEA, Tyrosinase, midkin, BAGE, CASP-8, β-catenin,β-catenin, γ-catenin, CA-125, CDK-1, CDK4, ESO-1, gp75, gp100, MART-1,MUC-1, MUM-1, p53, PAP, PSA, PSMA, ras, trp-1, HER-2, TRP-1, TRP-2,IL13Ralpha, IL13Ralpha2, AIM-2, AIM-3, NY-ESO-1, C9orf 112, SART1,SART2, SART3, BRAP, RTN4, GLEA2, TNKS2, KIAA0376, ING4, HSPH1, C13orf24,RBPSUH, C6orf153, NKTR, NSEP1, U2AF1L, CYNL2, TPR, SOX2, GOLGA, BMI1,COX-2, EGFRvIII, EZH2, LICAM, Livin, Livinβ, MRP-3, Nestin, OLIG2, ART1,ART4, B-cyclin, Gli1, Cav-1, cathepsin B, CD74, E-cadherin, EphA2/Eck,Fra-1/Fosl 1, GAGE-1, Ganglioside/GD2, GnT-V, β1,6-N, Ki67, Ku70/80,PROX1, PSCA, SOX10, SOX11, Survivin, UPAR, WT-1, Dipeptidyl peptidase IV(DPPIV), adenosine deaminase-binding protein (AD Abp), cyclophilin b,Colorectal associated antigen (CRC)-C017-1A/GA733, T-cellreceptor/CD3-zeta chain, GAGE-family of tumor antigens, RAGE, LAGE-I,NAG, GnT-V, RCAS1, α-fetoprotein, pl20ctn, Pmel117, PRAME, brainglycogen phosphorylase, SSX-I, SSX-2 (HOM-MEL-40), SSX-I, SSX-4, SSX-5,SCP-I, CT-7, cdc27, adenomatous polyposis coli protein (APC), fodrin,PlA, Connexin 37, Ig-idiotype, p15, GM2, GD2 gangliosides, Smad familyof tumor antigens, Imp-1, EBV-encoded nuclear antigen (EBNA)-I,UL16-binding protein-like transcript 1 (Mult1), RAE-1 proteins, H60,MICA, MICB, and c-erbB-2, or an immunogenic peptide thereof, andcombinations thereof.

In another embodiment, the antigen is a target of modified T-cells,e.g., CAR T-cells described above. In such embodiments, the antigen isCD19 or a fragment thereof or a variant thereof. In another embodiment,the antigen is CD22 or a fragment thereof or a variant thereof.

The aforementioned antigens may be combined with the scaffoldcompositions using any known methods, including covalent andnon-covalent interactions. Some of these methods have been outlinedabove in sections relating to fabricating the MSR-SLB scaffolds with thefunctional molecules of the invention. Examples of non-covalentinteractions include, for example, electrostatic interactions, van derWaals' interactions, π-effects, hydrophobic interactions, physicalinsertion etc. For example, full length transmembrane protein antigenscan be incorporated into the lipid bilayer via physical insertion usingroutine methods. See, Cymer et al., Journal of Molecular Biology, 427.5:999-1022, 2015 and U.S. Pat. No. 7,569,850, which are incorporated byreference herein.

The antigens may also be attached or tethered to scaffold compositionsvia covalent interactions. Methods for attaching antigens toscaffolds/surfaces are known in the art, e.g., surface absorption,physical immobilization, e.g., using a phase change to entrap thesubstance in the scaffold material. Alternatively, covalent coupling viaalkylating or acylating agents may be used to provide a stable,long-term presentation of an antigen on the scaffold in a definedconformation. Exemplary reagents and methods for covalently couplingpeptides/proteins to polymers are known in the art. See, for example,U.S. Pat. No. 6,001,395, which is incorporated herein by reference. Inother embodiments, the antigens are encapsulated into the scaffolds.Methods for encapsulating antigens into suitable scaffolds, e.g., PLGAmicrospheres, are known in the art. See, for example, U.S. Pat. No.6,913,767 and International Publication No. WO 1995/011010, thedisclosures of each of which are incorporated herein by reference.

The antigens may be formulated to interact with the immune cell viadirect binding or indirect binding. Types of direct binding include, forexample, engagement or coupling of the antigen with the cognatereceptor, e.g., T-cell receptor. Indirect binding may occur through theintermediacy of one or more secondary agents or cell-types. For example,the antigen may first bind to a B-cell or an antigen-presenting cell(APC), get processed (e.g., degraded) and presented on cell-surfacemajor-histocompatibility complexes (MHC), to which the target cellpopulation, e.g., T-cell, binds. Alternately, the antigen may recruitother intermediary cells that secrete various cytokines, growth factors,chemokines, etc., which in turn attract the target immune cellpopulation. Whatever the mechanism may be, the recited components act inconcert to manipulate or modify the immune cells.

The antigen may be derived from a cell lysate, a fractionated celllysate, freshly harvested cells, biological fluids (including blood,serum, ascites), tissue extracts, etc. In one embodiment, the antigensare derived from lysates of target cells to which the desired immunecells, e.g., T cells, bind. In these embodiments, the antigens are firstfractionated in the cell lysate prior to loading the scaffolds. Thelysates may be derived from a desired target tissue, e.g., an autoimmunedisease-specific cells obtained from primary tissues. Alternately, thelysates may be derived from cancer cells, e.g., individual cellsobtained from tumor samples or tissue cultures or tumor cells obtainedfrom biopsies histologies.

The scaffolds of the invention may also contain one or more recruitingagents. The recruiting agent may be an agent selected from the groupconsisting of a T-cell recruiting agent, a B-cell recruiting agent, adendritic cell recruiting agent, and a macrophage recruiting agent.

In one embodiment, the scaffolds contain T-cell recruiting agents.Non-limiting examples of T-cell recruiting agents include, e.g.,granulocyte macrophage-colony stimulating factor (GM-CSF), chemokine(C-C motif) ligand 21 (CCL-21), chemokine (C-C motif) ligand 19(CCL-19), or a FMS-like tyrosine kinase 3 (Flt-3) ligand,granulocyte-colony stimulating factor (G-CSF), IFNγ, a C-X-C Motifchemokine ligand (CXCL) selected from the group consisting of CXCL12 andCXCR4, or a fragment thereof, a variant thereof, or a combinationthereof. Other types of T-cell recruiting agents include, ligands forCCR5 and CXCR3 receptors for recruiting T helper type 1 (Th1) subset.The CCR5 ligands, CCL5 and macrophage inflammatory proteins (MIP-la),are known. Alternately, ligands for CCR3, CCR4, CCR8 and CXCR4 may beemployed for specific recruitment of the Th2 subset. A combination ofthe ligands may also be employed.

Various homologs of the aforementioned T-cell recruiting agents,including functional fragments thereof, or variants thereof, are knownin the art. Representative examples of homologs include related proteinsfrom fly, mouse, rat, pig, cow, monkey, humans or the like. The homologspreferably include human or mouse homologs of the aforementionedrecruiting agents.

The scaffolds of the instant invention are adapted for the preferentialrecruitment of a single type or single sub-type of cell, for example,preferential recruitment of T-cells and particularly a subset of Tregcells or NK cells. Preferential recruitment is characterized by anaccumulation of at least 10%, at least 20%, at least 30%, at least 50%,at least 75%, at least 100%, at least 2-fold, at least 5-fold, at least8-fold, at least 10-fold, or greater increase in one or more of aparticular type of immune cells (e.g., T cells, B-cells, DC/macrophages)in the device compared to other types of immune cells in the device (orin control scaffolds that are devoid of recruitment agents). Inscaffolds that are adapted to recruit a combination of immune cells,e.g., a combination of T-cells and DC/macrophages, preferentialrecruitment is characterized where the total percentage of recruitedcells is at least 10%, at least 20%, at least 30%, at least 50%, atleast 75%, at least 100%, at least 2-fold (i.e., 200%), at least 5-fold,at least 8-fold, at least 10-fold, or greater than other types of immunecells in the device (or in control scaffolds). Particularly,preferential recruitment is characterized by 1-10 fold increase in thenumber of the cells of interest compared to other immune cells.

In one embodiment, the instant invention relates to MSR-SLB scaffoldsfurther comprising a recruitment agent which is GM-CSF, an agonistthereof, a mimetic thereof, a fragment thereof, a variant thereof, or acombination thereof. Preferably, the recruitment agent is GM-CSF incombination with at least one of CCL-21, CCL-19, Flt-3 or GCSF.Representative examples of such recruitment agents include, e.g., humanGM-CSF (NCBI Accession #NP_000749.2) and mouse GM-CSF (NCBI Accession#NP_034099.2). In another embodiment, the instant invention relates toMSR-SLB scaffolds containing fragments of GM-CSF, e.g., a polypeptidecontaining amino acids 18-144 of the hGM-CSF sequence. In yet anotherembodiment, the invention relates to scaffolds containing GM-CSFvariants including, for example, VAR_013089 and VAR_001975, thesequences of which have been accessioned in UNIPROT (Accession No.P04141). In another embodiment, the invention relates to MSR-SLBscaffolds containing GM-CSF mimetics including, for example, antibodiesbinding to GM-CSF receptor, e.g., those described by Monfardini et al.,Curr Pharm Des., 8(24): 2185-99, 2002.

Embodiments of the invention further provide for scaffolds formanipulating immune cells which comprise a plurality of additionalagents. In such embodiments, the additional agent may comprise a growthfactor, a cytokine, a chemokine, an interleukin, an adhesion signalingmolecule, an integrin signaling molecule or a fragment thereof or acombination thereof.

Representative examples of growth factors/cytokines include, but are notlimited to, adrenomedullin (AM), angiopoietin (Ang), autocrine motilityfactor, bone morphogenetic proteins (BMPs), brain-derived neurotrophicfactor (BDNF), epidermal growth factor (EGF), erythropoietin (EPO),fibroblast growth factor (FGF), foetal Bovine Somatotrophin (FBS) glialcell line-derived neurotrophic factor (GDNF), granulocytecolony-stimulating factor (G-CSF), granulocyte macrophagecolony-stimulating factor (GM-CSF), growth differentiation factor-9(GDF9), hepatocyte growth factor (HGF), hepatoma-derived growth factor(HDGF), insulin-like growth factor (IGF), keratinocyte growth factor(KGF), migration-stimulating factor (MSF), myostatin (GDF-8), nervegrowth factor (NGF), neurotrophins, platelet-derived growth factor(PDGF), thrombopoietin (TPO), T-cell growth factor (TCGF), transforminggrowth factor (TGF-α or TGF-β), tumor necrosis factor-alpha (TNF-α),vascular endothelial growth factor (VEGF), Wnt, placental growth factor(PGF), or functional fragment thereof, or a combination thereof.

Representative types of interleukins include, but are not limited to,IL-1 (activates T cells, B-cells, NK cells, and macrophages), IL-2(activates B-cells and NK cells), IL-3 (stimulates non-lymphoid cells),IL-4 (growth factor for activated B cells, resting T cells, and mastcells), IL-5 (for differentiation of activated B cells), IL-6 (growthfactor for plasma cells and T-cells), IL-7 (growth factor for preB-cells/pre T-cells and NK cells), IL-10 (activates macrophages,B-cells, mast cells, Th1/Th2 cells), IL-12 (activates T cells and NKcells), IL-17 (activates Th cells). Functional fragments ofinterleukins, which are characterized by their ability to modulate theactivity of target cells, may also be employed.

Optionally, the scaffolds may contain adhesion molecules, which may alsoserve as signaling agents. Representative examples of adhesion signalingmolecules include, but are not limited to, fibronectin, laminin,collagen, thrombospondin 1, vitronectin, elastin, tenascin, aggrecan,agrin, bone sialoprotein, cartilage matrix protein, fibronogen, fibrin,fibulin, mucins, entactin, osteopontin, plasminogen, restrictin,serglycin, SPARC/osteonectin, versican, von Willebrand Factor,polysaccharide heparin sulfate, connexins, collagen, RGD (Arg-Gly-Asp)and YIGSR (Tyr-Ile-Gly-Ser-Arg) peptides and cyclic peptides,glycosaminoglycans (GAGs), hyaluronic acid (HA), condroitin-6-sulfate,integrin ligands, selectins, cadherins and members of the immunoglobulinsuperfamily. Other examples include neural cell adhesion molecules(NCAMs), intercellular adhesion molecules (ICAMs), vascular celladhesion molecule (VCAM-1), platelet-endothelial cell adhesion molecule(PECAM-1), L1, and CHL1. Functional fragments of the adhesion molecules,which are characterized by their ability to modulate the binding oftarget cells to the scaffolds of the invention, may also be employed.Particularly, adhesion molecules comprise peptides or cyclic peptidescontaining the amino acid sequence arginine-glycine-aspartic acid (RGD),which is known as a cell attachment ligand and found in various naturalextracellular matrix molecules. In another embodiment, the adhesionpeptide is a collagen mimic. Representative examples include, thepeptide having the structure GGYGGGPC(GPP)5GFOGER(GPP)5GPC, wherein O ishydroxyproline. Such peptides may be collectively referred to as GFOGERpeptides. GFOGER peptides have been previously shown to be particularlygood for T cell adhesion. See, Stephan et al, Nature Biotechnology 33,2015.

A polymer matrix with such a modification provides cell adhesionproperties to the scaffold of the invention, and sustains long-termsurvival of mammalian cell systems, as well as supporting cell growthand differentiation. The adhesion molecules may be coupled to thepolymer matrix is accomplished using synthetic methods which are ingeneral known to one of ordinary skill in the art and are described inthe examples. See, e.g., Hirano et al., Advanced Materials, 17-25, 2004;Hermanson et al., Bioconjugate Techniques, p. 152-185, 1996; Massia andHubbell, J. Cell Biol. 114:1089-1100, 1991; Mooney et al., J. Cell Phys.151:497-505, 1992; and Hansen et al., Mol. Biol. Cell 5:967-975, 1994,the disclosures in which are incorporated by reference.

Depending on the target cell type, it may be preferable to employadhesion signaling molecules that are specific for the target cells.Thus, in one embodiment, the scaffolds contain adhesion receptors thatare useful in the binding/sequestration of T-cells. In theseembodiments, the scaffolds may contain T-cell specific adhesionmolecules, for example, a receptor selected from the group consisting ofMHC class II (for CD4+ cells), MHC class I (for CD8+ cells), LFA-3 (CD2ligand), ICAM1 (ligand for LFA-1) or a variant thereof, a fragmentthereof or a combination thereof.

Depending on need, the scaffolds may be specifically formulated tocontain a subset of recruitment agents and adhesion molecules so as tomanipulate a particular subset of immune cells, e.g., a particularsub-population of T-cells. In these embodiments, the scaffolds may beformulated/fabricated using agents that specifically bind tocell-surface markers that are expressed in the target cells. Forexample, in the context of T-cells, the scaffolds may be adapted for thepreferential recruitment of helper T-cells (T_(H) cells; whichdifferentially express CD4+), cytotoxic T-cells (T_(c) cells; whichdifferentially express CD8+), memory T-cells (Tm cells; whichdifferentially express CD45RO), suppressor T-cells (T_(s) which cells),regulatory T-cells (Tregs; further characterized as FOXP3+ Treg cellsand FOXP3-Treg), natural killer T-cells (NK cells; differentiallyexpress CD1d+), mucosal associated invariant (MAITs; differentiallyexpress MR1), gamma delta T cells, (γδ T cells; comprise TCRs containingone γ-chain and one 6-chain). Such agents which bind to cell-surfacemarkers may include, for example, haptens, peptides, ligands,antibodies, or the like. Other routine techniques for enriching theisolates with one or more cell subtype may be optionally used in situ orex situ.

The scaffolds may also be adapted for recruiting immune cells that arespecific for a disease. For example, a plurality of T cells that arespecific for a particular type of autoimmune disease may be recruited.Thus in one embodiment, scaffolds that are useful in the diagnosis ofautoimmune disorders may be formulated to contain recruitment agentsthat are specific to the immune cells implicated in the disorder. Suchrecruitment agents may, for example, be specific to regulatory T cells(Tregs), suppressor T cells (T_(s)) or a combination thereof. In arelated embodiment, scaffolds that are useful in the diagnosis ofcancers may be formulated to contain recruitment agents forpreferentially recruiting cancer-specific T-cell types, e.g., cytotoxicT cells (Tc), natural killer cells (NK) or a combination thereof.

In certain embodiments, the scaffold is useful to pan fordisease-specific cells. Such may include, for example, cells thatdirectly promote disease progression. In the context of many autoimmunediseases, the disease may mediate and promote via targeted killing ofspecific population of cells, e.g., beta cells of pancreas in T1D andneuronal cells in multiple sclerosis. In other autoimmune diseases, thedisease may be precipitated by targeted attack of specific epitopes suchas, for example, rheumatoid factors (RF) and citrullinated peptides(ACPA) in the context of rheumatoid arthritis and antigens present inthe gut flora in the context of Crohn's disease. The targeteddestruction of the cells generally involves specific type or subset ofimmune cells. Thus, based on the nature and properties of the cellulartargets, immune cells that are specific thereto may be preferentiallymanipulated using the scaffolds of the instant invention.

In the aforementioned embodiments, the scaffolds are provided withantigens to which disease-specific immune cells, e.g., T cells, bind.These autoimmune cells can be manipulated and optionally re-programmedto a non-autoimmune phenotype. Methods of reprogramming T-cells topluripotency are known in the art. See, Nishimura et al., Stem Cell 12,114-126 (2013); Themeli et al., Nature Biotechnology 31, 928-933 (2013).In certain instances, particularly in the context of cancer-specificT-cells, the reprogrammed cells may be rejuvenated to target the cancer.Alternately, in the context of T-cells that are specific to autoimmunediseases, the cells may be eliminated.

In certain embodiments, the scaffold of the invention are fabricated asporous structures that have been engineered to sustain antigenpresentation. Methods for fabricating porous scaffolds have beendescribed in the art. See, for example, U.S. Publication Nos.2011/0020216, 2013/0202707, 2011/0020216 and U.S. Pat. No. 8,067,237,the disclosures in which are incorporated by reference herein.

Embodiments of the invention further provide for scaffolds containingMSR-SLB scaffolds that possess the desired stability for various ex vivoand in vivo applications. For example, the scaffolds are stable intissue culture applications, cell growth experiments, or as transplantmaterial to be administered into tissues (harvested or engineered) andalso into subjects. In one embodiment, the invention relates tomesoporous silica microrod-lipid bilayer (MSR-SLB) scaffolds whichretain a continuous, fluid architecture for at least 0.5 days, 1 day, 2days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days,11 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days, 18 days,19 days, 20 days, 21 days, 25 days, 30 days, 35 days, 40 days, 45 days,50 days, or more. The stability and/or fluid architecture of thescaffolds may be monitored using routine techniques, e.g., themicroscopic visualization techniques illustrated in the Examples below.

II. Methods of Making the Scaffolds of the Invention

Embodiments of the invention further relate to methods for making theantigen-presenting cell mimetic scaffolds (APC-MS) of the invention. Themethod comprises providing a base layer comprising high surface areamesoporous silica micro-rods (MSR); optionally loading the T-cellhomeostatic agents on the MSR; layering a continuous, fluid supportedlipid bilayer (SLB) on the base layer comprising the MSRs, therebygenerating an MSR-SLB scaffold; loading the T-cell homeostatic agents onthe MSR-SLB scaffold if step (b) is not carried out; optionally blockingone or more non-specific integration sites in the MSR-SLB scaffold witha blocker; and loading the T-cell activating molecules and the T-cellco-stimulatory molecules onto the MSR-SLB scaffold, thereby making theAPC-MS. In these embodiments, the method(s) may include further loadingat least one additional agent which is a growth factor, a cytokine, aninterleukin, an adhesion signaling molecule, an integrin signalingmolecule, or a fragment thereof or a combination thereof in thescaffold. Methods for loading the additional ingredients have beendescribed previously in the device fabrication section. A representativemethod for making the scaffolds of the invention is provided in FIG. 24.

In one embodiment, a mixture of functional molecules containing a 1:1mixture of the T-cell activating molecules and the T-cell co-stimulatorymolecules (e.g., anti-CD3 antibody and anti-CD28 antibody) is combinedwith the MSR-SLB scaffold such that the weight ratio of the functionalmolecules:MSR-SLB scaffold is between about 1:2 and about 1:20,preferably between about 1:4 and about 1:15, a particularly betweenabout 1:5 to about 1:10. The weight ratio of the T-cell activatingmolecule and the T-cell co-stimulatory molecule may be adjusted, e.g.,between about 5:1 to about 1:5, while retaining the same dry weightratio between the functional molecules and the MSR-SLB scaffold.

Furthermore, embodiments of the invention further relate to methods ofmaking the APC-MS by assembling a plurality of scaffolds to generatestacks with sufficient porosity to permit infiltration of T cells, morespecifically, distinct sub-populations of helper T-cells or cytotoxicT-cells.

III. Methods for Using the Scaffolds of the Invention

The scaffolds of the invention may be used for various applications,including, but not limited to, manipulation of target effector cells,e.g., T-cells, isolation of a specific population of effector cells,e.g., a sub-population of CD8+ T-cells, diagnosis and therapy ofdiseases, and the production of compositions and kits for the diagnosisand therapy of diseases.

Methods for the Manipulation of Target Cells

In one embodiment, the instant invention provides a method formanipulating target effector cells or a sub-population thereof (e.g.,helper T-cells or cytotoxic T-cells). In this context, the term“manipulation” includes, for example, activation, division,differentiation, growth, expansion, reprogramming, anergy, quiescence,senescence, apoptosis or death of the target effector cells.

In one embodiment, the target effector cells, e.g., T-cells, aremanipulated (e.g., activated) in situ by providing scaffolds of theinvention such that the target effector cells come into contact with thescaffolds. In order to facilitate the contact, the scaffolds may beimplanted at a suitable site in a subject, e.g., subcutaneously orintravenously. In other embodiments, the target cells are manipulated exvivo by culturing a sample containing target effector cells with thescaffolds of the invention.

A variety of target effector cells may be manipulated, including, freshsamples employed from subjects, primary cultured cells, immortalizedcells, cell-lines, hybridomas, etc. The manipulated cells may be usedfor various immunotherapeutic applications as well as for research.

The site of manipulation of target effector cells may be in situ or exsitu. Thus, in one embodiment, the cells are manipulated in situ (e.g.,within the scaffold). In this context, the cells need not be physicallyremoved from the scaffold to be manipulated. In another embodiment, thecells are manipulated ex situ (e.g., by first removing the cells fromthe scaffold and manipulating the removed cells). When the scaffolds areimplanted into a subject, the cells may be manipulated at or near theimplant site. In other embodiments, the implanted scaffolds may be firstremoved from the implant site and the effector cells may be manipulatedin situ or ex situ, as described previously.

In certain embodiments, the scaffolds used in manipulating effectorcells may be provided with antigen presenting cells (APC) and/or variousantigens derived from such APCs. These secondary agents (e.g., APCs orantigens derived from APCs) may be provided in the scaffold structure orprovided extrinsically, e.g., in culture media. In certain embodiments,the scaffolds may be provided with various antigens that attract and/orrecruit APCs. Representative examples of such attracting and/orrecruiting molecules have been provided in the previous sections.

In certain embodiments, the antigen-containing scaffolds may be used tomanipulate target effector cells in vivo. For such applications, thescaffolds may be implanted inside a blood vessel, in the lympatictissue, at the tumor site, at a disease site (e.g., areas surroundingtissues affected by rheumatoid arthritis) or subcutaneously, such thatthe target effector cells come into contact with the scaffolds.Alternately, the scaffolds may be injected in a minimally invasivemanner, for example, via needle, catheter or the like. The implantedscaffolds may be allowed to remain at the implant site for about 0.5day, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 1 week, 2 weeks, 3weeks, 4 weeks, 1 month, 2 months, 3, months, 6 months, 7, months, 8months, 9 months. 1 year, 2 years, or more. Periodically, the scaffoldsmay be explanted to study, analyze, or even further manipulate theeffector cells.

In a related embodiment, the instant invention relates to manipulationof antigen-specific effector cells in situ. In this context, thescaffolds of the present invention may contain antigens of interestwhich are adsorbed onto the scaffold using the same strategies foradsorbing the functional molecules. Alternately, the scaffolds of theinvention may be incubated with a sample containing the antigen-specificeffector T-cells in culture media together with APCs that display theantigen of interest. The target effector cells are then allowed to comeinto contact with the scaffolds and the functional molecules containedin the scaffolds act together to promote the manipulation of effectorcells. Purely as a representative embodiment, as described in theExamples section, a sample containing T cells is incubated with thescaffolds of the present invention, which activates, co-stimulates andhomeostatically maintains the target effector cells. The sample may beincubated with the scaffold for about 1 day to 30 days, for about 1 to15 days or for about 4 to 13 days, e.g., for about 7-8 days, resultingin the selective manipulation of the effector cell population. Theantigen-specific effector cells may be additionally manipulated byselecting cells based on the expression of certain gene products, e.g.,T-cell receptors (TCR) that recognize the antigen or theantigen-presenting cells of interest.

Embodiments described herein further relate to methods for manipulatingantigen-specific effector cells ex situ, wherein the scaffolds areprovided with the APC expressing the antigen of interest or the antigenitself. The manipulation step may be carried out ex situ or in situ.

In another embodiment, the effector target cells which are specific tothe antigen or APCs may be selectively manipulated over other effectorcells (e.g., favoring CD8+ T-cells over CD4+ T-cells). For example, asample containing CD8+ T-cells (along with CD4+ T-cells) may beincubated with the scaffolds of the present invention which aremechanically or chemically fabricated to permit infiltration and/orsequestration of CD8+ T-cells. The infiltrated and/or sequestered CD8+T-cells may be further expanded, activated, proliferated, or grown usingtechniques known in the art. Representative methods have been describedpreviously.

In another embodiment, the effector target cells which are specific tothe antigen or APCs may be undesired (e.g., regulatory/suppressorT-cells) and they are induced to undergo apoptosis, anergy or deathfollowing contact with the scaffolds of the instant invention. Forexample, a sample containing regulatory T cells (along with otherT-cells) may be incubated with the scaffolds of the present inventionwhich are mechanically or chemically fabricated to permit infiltrationand/or sequestration of regulatory/suppressor T-cells. The infiltratedand/or sequestered T-cells may be eliminated using techniques known inthe art.

In this context, the identity of the cells that have infiltrated and/orare sequestered in the scaffolds of the invention may be furtherdetermined using art-known techniques. Thus, in one embodiment, the geneproduct for identifying or selecting for activated T cells may be a cellsurface marker or cytokine, or a combination thereof. Cell surfacemarkers for identifying activated T cells include, but are not limitedto, CD69, CD4, CD8, CD25, HLA-DR, CD28, and CD134. CD69 is an earlyactivation marker found on B and T lymphocytes, NK cells andgranulocytes. CD25 is an IL-2 receptor and is a marker for activated Tcells and B cells. CD4 is a TCR coreceptor and is marker for thymoctes,TH1- and TH2-type T cells, monocytes, and macrophages. CD8 is also a TCRcoreceptor and is marker for cytotoxic T cells. CD134 is expressed onlyin activated CD4+ T cells.

Cell surface markers for selecting for activated T cells include, butare not limited to, CD36, CD40, and CD44. CD28 acts as a stimulatoryT-cell activation pathway independent of the T-cell receptor pathway andis expressed on CD4+ and CD8+ cells. CD3δ is a membrane glycoprotein andis a marker for platelets, monocytes and endothelial cells. CD40 is amarker for B cells, macrophages and dendritic cells. CD44 is a markerfor macrophages and other phagocytic cells. Subsets of T cells may beisolated by using positive selection, negative selection, or acombination thereof for expression of cell surface gene products ofhelper T cells or cytotoxic T cells (e.g., CD4 vs. CD8). Cytokines foridentifying activated T cells of the present invention include, but arenot limited to cytokines produced by TH1-type T cells (cell-mediatedresponse) and TH2-type T cells (antibody response). Cytokines foridentifying activated TH1-type T cells include, but are not limited to,IL-2, gamma interferon (IFN-γ) and tissue necrosis factor alpha (TNFα).Cytokines for identifying activated TH2-type T cells include, but notlimited to, IL-4, IL-5, IL-10 and IL-13. Subsets of T cells may also beisolated by using positive selection, negative selection, or acombination thereof for expression of cytokine gene products of helper Tcells or cytotoxic T cells (e.g., IFN-γ vs. IL4).

An activated TH1-type T cell specific for an antigen of interest may beisolated by identifying cells that express CD69, CD4, CD25, IL-2, IFNγ,TNFα, or a combination thereof. An activated TH1-type T cell specificfor an antigen of interest may also be isolated by identifying cellsthat express CD69 and CD4 together with IFNγ or TNFα. An activatedTH2-type T cell specific for an antigen of interest may be isolated byidentifying cells that express CD69, CD4, IL-4, IL-5, IL-10, IL-13, or acombination thereof. A combination of an activated TH1-type T cell and aTH2-type T cell specific for an antigen of interest may be isolated byidentifying cells that express CD69, CD4, CD25, IL-2, IFNγ, TNFα, or acombination thereof and cells that express CD69, CD4, IL-4, IL-5, IL-10,IL-13, or a combination thereof.

The gene products used for positive or negative selection of theactivated T cells of the present invention may be identified byimmunoselection techniques known to those in the art which utilizeantibodies including, but not limited to, fluorescence activated cellsorting (FACS), magnetic cell sorting, panning, and chromatography.Immunoselection of two or more markers on activated T cells may beperformed in one or more steps, wherein each step positively ornegatively selects for one or more markers. When immunoselection of twoor more markers is performed in one step using FACS, the two or moredifferent antibodies may be labeled with different fluorophores.Alternately, as described above, cells may be sorted using microbeads.

For cell-surface expressed gene products, the antibody may directly bindto the gene product and may be used for cell selection. For cell-surfacegene products expressed at low concentrations, magnetofluorescentliposomes may be used for cell selection. At low levels of expression,conventional fluorescently labeled antibodies may not be sensitiveenough to detect the presence of the cell surface expressed geneproduct. Fluorophore-containing liposomes may be conjugated toantibodies with the specificity of interest, thereby allowing detectionof the cell surface markers.

For intracellular gene products, such as cytokines, the antibody may beused after permeabilizing the cells. Alternatively, to avoid killing thecells by permeabilization, the intracellular gene product if it isultimately secreted from the cell may be detected as it is secretedthrough the cell membrane using a “catch” antibody on the cell surface.The catch antibody may be a double antibody that is specific for twodifferent antigens: (i) the secreted gene product of interest and (ii) acell surface protein. The cell surface protein may be any surface markerpresent on T cells, in particular, or lymphocytes, in general, (e.g.,CD45). The catch antibody may first bind to the cell surface protein andthen bind to the intracellular gene product of interest as it issecreted through the membrane, thereby retaining the gene product on thecell surface. A labeled antibody specific for the captured gene productmay then be used to bind to the captured gene product, which allows theselection of the activated T cell. Certain forms of cytokines are alsofound expressed at low concentration on the cell surface. For example,IFN-γ is displayed at a low concentration on the cell surface withkinetics similar to those of intracellular IFN-γ expression(Assenmacher, et al. Eur J. Immunol, 1996, 26:263-267). For forms ofcytokines expressed on the cell surface, conventional fluorescentlylabeled antibodies or fluorophore containing liposomes may be used fordetecting the cytokine of interest. One of ordinary skill in the artwill recognize other techniques for detecting and selectingextracellular and intracellular gene products specific for activated Tcells.

The T cells isolated by the methods of the present invention may beenriched by at least 40%-90% from whole blood. The T cells may also beenriched by at least 95% from whole blood. The T cells may also beenriched by at least 98% from whole blood. The T cells may also beenriched at least 99.5% from whole blood. Similar methods may be used inthe in situ or ex situ manipulation of B-cells. In certain embodiments,cryopreserved cells are thawed and washed as described herein andallowed to rest for one hour at room temperature prior to activation.

Depending upon application, the dry weight ratios of scaffolds to cellsample may be adjusted. For example, the scaffold: cell dry weight ratiomay range from 1:500 to 500:1 and any integer values in between may beused to manipulate effector cells. As those of ordinary skill in the artcan readily appreciate, the ratio of scaffold to cells may dependent onthe scaffold size relative to the target cell.

Expansion of T Cell Population

In a related embodiment, the present invention further relates tomethods for expanding T-cells from a population of immune cells, e.g.,expanding T-cells contained in sample containing B-cells, dendriticcells, macrophages, plasma cells, and the like. In another embodiment,the present invention also relates to methods for expanding a specificpopulation of T-cells, e.g., expanding cytotoxic T-cells from a samplecontaining helper T-cells, natural killer T-cells, regulatory/suppressorT-cells, and the like. The specific sub-population of T-cells may beused downstream in various immunotherapeutic applications. Withoutwishing to be bound by any particular theory, it is believed that theAPC-MS of the instant invention are particularly effective for theexpansion of T-cells because the relatively large size and high aspectratio of the mesoporous silica rods allow for the formation of largeclusters of T-cells interacting with each rod which may promote theeffective expansion of T-cells by allowing T-cell/T-cell interactionsand/or paracrine signaling.

In one embodiment, the target effector cells, e.g., T-cells, areexpanded (e.g., grown or differentiated) in situ by providing scaffoldsof the invention such that the target effector cells come into contactwith the scaffolds. In order to facilitate the contact, the scaffoldsmay be implanted at a suitable site in a subject, e.g., subcutaneouslyor intravenously. In other embodiments, the target cells are expanded exvivo by culturing a sample containing target effector cells with thescaffolds of the invention. In one embodiment, ex vivo T cell expansioncan be performed by first isolating T-cells from a sample andsubsequently stimulating T-cells by contacting with the scaffolds of theinvention, such that, the effector T-cells are activated, co-stimulatedand homeostatically maintained.

In one embodiment of the invention, the T cells are primary T-cellsobtained from a subject. The term “subject” is intended to includeliving organisms in which an immune response can be elicited (e.g.,mammals). Examples of subjects include humans, dogs, cats, mice, rats,and transgenic species thereof. T-cells can be obtained from a number ofsources, including peripheral blood mononuclear cells, bone marrow,lymph node tissue, cord blood, thymus tissue, tissue from a site ofinfection, spleen tissue, and tumors. In certain embodiments of thepresent invention, any number of primary T-cells and/or T-cell linesavailable in the art, may be used.

Studies on whole blood counts reveal that the number of T-cells in wholeblood is very low. For example, according to the product catalogpublished by Stem Cell Technologies, Vancouver, BC, CANADA (Document#23629, VERSION 2.1.0), the leukocyte population in whole blood is about0.1-0.2% (due to predominance of erythrocytes), of which T-cells make upabout 7-24% of the overall leukocyte population. Among T-cells, CD4+T-cells make up about 4-20% of the overall leukocyte population(translating to less than 0.04% of the overall cell population in wholeblood) and CD8+ T-cells make up about 2-11% of the overall leukocytepopulation (translating to less than 0.022% of the overall cellpopulation in whole blood). Thus, in certain embodiments of the presentinvention, methods of the invention may be coupled with other art-knowntechniques for enrichment of target cells. The enrichment step may becarried out prior to contacting the sample with the scaffolds of theinstant invention. In another embodiment, the enrichment step may becarried out after the sample has been contacted with the scaffolds ofthe present invention.

In one embodiment, the effector cell population may be enriched usingFICOLL separation. In one embodiment, cells from the circulating bloodof an individual are obtained by apheresis or leukapheresis. Theapheresis product typically contains lymphocytes, including T cells,monocytes, granulocytes, B cells, other nucleated white blood cells, redblood cells, and platelets. The cells collected by apheresis may bewashed to remove the plasma fraction and to place the cells in anappropriate buffer or media for subsequent processing steps. The cellsare then washed with phosphate buffered saline (PBS). Alternately, thewash solution lacks calcium and may lack magnesium or may lack many ifnot all divalent cations. A semi-automated “flow-through” centrifuge mayalso be used according to the manufacturer's instructions. Afterwashing, the cells may be resuspended in a variety of biocompatiblebuffers, such as, for example, Ca-free, Mg-free PBS. Alternatively, theundesirable components of the apheresis sample may be removed and thecells directly resuspended in culture media.

In another embodiment, peripheral or whole blood T cells may be enrichedby lysing the red blood cells and depleting the monocytes, for example,by centrifugation through a PERCOLL™ gradient. A specific subpopulationof T cells, such as CD28+, CD4+, CD8+, CD45RA+, and CD45RO+T cells, canbe further isolated by positive or negative selection techniques.

In accordance with the present invention, various sorting techniques maybe optionally employed. For example, the expanded or manipulated T cellpopulation may be further sorted using a combination of antibodiesdirected to surface markers unique to the cells. A preferred method iscell sorting and/or selection via magnetic immunoadherence or flowcytometry that uses a cocktail of monoclonal antibodies directed to cellsurface markers present on the cells selected. For example, to enrichfor CD4+ cells by negative selection, a monoclonal antibody cocktailtypically includes antibodies to CD14, CD20, CD11b, CD16, HLA-DR, andCD8. In certain embodiments, it may be desirable to enrich or negativelyselect regulatory T cells which typically express CD4+, CD25+, CD62Lhi,GITR+, and FoxP3+.

For isolation of a desired population of cells, the concentration ofcells and scaffold surface can be varied. In certain embodiments, it maybe desirable to significantly decrease the volume in which the scaffoldsand cells are mixed together (i.e., increase the concentration ofcells), to ensure maximum contact of cells and scaffolds. For example,in one embodiment, a concentration of 2 billion cells/ml is used. In oneembodiment, a concentration of 1 billion cells/ml is used. In a furtherembodiment, greater than 100 million cells/ml is used. In a furtherembodiment, a concentration of cells of 10, 15, 20, 25, 30, 35, 40, 45,or 50 million cells/ml is used. In yet another embodiment, aconcentration of cells from 75, 80, 85, 90, 95, or 100 million cells/mlis used. In further embodiments, concentrations of 125 or 150 millioncells/ml can be used. Using high concentrations can result in increasedcell yield, cell activation, and cell expansion. Further, use of highcell concentrations allows more efficient capture of cells that mayweakly express target antigens of interest, such as CD28-negative Tcells, or from samples where there are many tumor cells present (i.e.,leukemic blood, tumor tissue, etc.). Such populations of cells may havetherapeutic value and would be desirable to obtain. For example, usinghigh concentration of cells allows more efficient selection of CD8+ Tcells that normally have weaker CD28 expression.

In a related embodiment, it may be desirable to use lower concentrationsof cells. This can be achieved by lowering the scaffold:cell ratio, suchthat interactions between the scaffolds and cells are minimized. Thismethod selects for cells that express high amounts of desired antigensto be bound to the scaffolds. For example, CD4+ T cells express higherlevels of CD28 and are more efficiently captured than CD8+ T cells indilute concentrations. In one embodiment, the concentration of cellsused is 5×10⁶/ml. In other embodiments, the concentration used can befrom about 1×10⁵/ml to 1×10⁹/ml, and any integer value in between, e.g.,1×10⁵/ml to 1×10⁸/ml, 1×10⁶/ml to 1×10⁷/ml, 1×10⁷/ml to 1×10⁹/ml.

In one embodiment, the instant invention may include art-knownprocedures for sample preparation. For example, T cells may be frozenafter the washing step and thawed prior to use. Freezing and subsequentthawing provides a more uniform product by removing granulocytes and tosome extent monocytes in the cell population. After the washing stepthat removes plasma and platelets, the cells may be suspended in afreezing solution. While many freezing solutions and parameters areknown in the art and will be useful in this context, one method involvesusing PBS containing 20% DMSO and 8% human serum albumin, or othersuitable cell freezing media containing for example, HESPAN andPLASMALYTE A, the cells then are frozen to −80° C. at a rate of 1° perminute and stored in the vapor phase of a liquid nitrogen storage tank.Other methods of controlled freezing may be used as well as uncontrolledfreezing immediately at −20° C. or in liquid nitrogen.

Also contemplated in the context of the invention is the collection ofblood samples or leukapheresis product from a subject at a time periodprior to when the expanded cells as described herein might be needed. Assuch, the source of the cells to be expanded can be collected at anytime point necessary, and desired cells, such as T cells, isolated andfrozen for later use in T cell therapy for any number of diseases orconditions that would benefit from T cell therapy, such as thosedescribed herein. In one embodiment a blood sample or a leukapheresis istaken from a generally healthy subject. In certain embodiments, a bloodsample or a leukapheresis is taken from a generally healthy subject whois at risk of developing a disease, but who has not yet developed adisease, and the cells of interest are isolated and frozen for lateruse. In certain embodiments, the T cells may be expanded, frozen, andused at a later time. In certain embodiments, samples are collected froma patient shortly after diagnosis of a particular disease as describedherein but prior to any treatments. In a further embodiment, the cellsare isolated from a blood sample or a leukapheresis from a subject priorto any number of relevant treatment modalities, including but notlimited to treatment with agents such as antiviral agents, chemotherapy,radiation, immunosuppressive agents, such as cyclosporin, azathioprine,methotrexate, mycophenolate, and FK506, antibodies, or otherimmunoablative agents such as CAMPATH, anti-CD3 antibodies, cytoxin,fludaribine, cyclosporin, FK506, rapamycin, mycophenolic acid, steroids,FR901228, and irradiation. These drugs inhibit either the calciumdependent phosphatase calcineurin (cyclosporine and FK506) or inhibitthe p70S6 kinase that is important for growth factor induced signaling(rapamycin). (Liu et al., Cell 66:807-815, 1991; Henderson et al.,Immun. 73:316-321, 1991; Bierer et al., Curr. Opin. Immun. 5:763-773,1993; Isoniemi (supra)). In a further embodiment, the cells are isolatedfor a patient and frozen for later use in conjunction with (e.g. before,simultaneously or following) bone marrow transplantation, T cellablative therapy using either chemotherapy agents such as, fludarabine,external-beam radiation therapy (XRT), cyclophosphamide, or antibodiessuch as OKT3 or CAMPATH. In another embodiment, the cells are isolatedprior to and can be frozen for later use for treatment following B-cellablative therapy such as agents that react with CD20, e.g. Rituxan.

In a further embodiment of the present invention, T cells are obtainedfrom a patient directly following treatment. In this regard, it has beenobserved that following certain cancer treatments, in particulartreatments with drugs that damage the immune system, shortly aftertreatment during the period when patients would normally be recoveringfrom the treatment, the quality of T cells obtained may be optimal orimproved for their ability to expand ex vivo. Likewise, following exvivo manipulation using the methods described herein, these cells may bein a preferred state for enhanced engraftment and in vivo expansion.Thus, it is contemplated within the context of the present invention tocollect blood cells, including T cells, dendritic cells, or other cellsof the hematopoietic lineage, during this recovery phase. Further, incertain embodiments, mobilization (for example, mobilization withGM-CSF) and conditioning regimens can be used to create a condition in asubject wherein repopulation, recirculation, regeneration, and/orexpansion of particular cell types is favored, especially during adefined window of time following therapy. Illustrative cell typesinclude T cells, B cells, dendritic cells, and other cells of the immunesystem.

Scaffolds containing any ratio of T-cell activating molecules:T-cellco-stimulatory molecules may be used in accordance with the presentmethods. In one embodiment, wherein the T-cell activating molecule andthe T-cell co-stimulatory molecules are both antibodies, a 1:1 ratio ofeach antibody may be used. In one embodiment, the ratio of CD3:CD28antibody bound to the scaffolds ranges from 100:1 to 1:100 and allinteger values there between. In one aspect of the present invention,more anti-CD28 antibody is bound to the scaffolds than anti-CD3antibody, i.e. the ratio of CD3:CD28 is less than one. In certainembodiments of the invention, the ratio of anti CD28 antibody to antiCD3 antibody bound to the scaffolds is greater than 2:1. In oneparticular embodiment, a 1:100 CD3:CD28 ratio of antibody bound toscaffolds is used. In another embodiment, a 1:75 CD3:CD28 ratio ofantibody bound to scaffolds is used. In a further embodiment, a 1:50CD3:CD28 ratio of antibody bound to scaffolds is used. In anotherembodiment, a 1:30 CD3:CD28 ratio of antibody bound to scaffolds isused. In one preferred embodiment, a 1:10 CD3:CD28 ratio of antibodybound to scaffolds is used. In another embodiment, a 1:3 CD3:CD28 ratioof antibody bound to the scaffolds is used. In yet another embodiment, a3:1 CD3:CD28 ratio of antibody bound to the scaffolds is used.

One aspect of the present invention stems from the surprising findingthat wherein the method confers increased expansion of the population ofT-cells after about 1 week of contact with the scaffold compared to acontrol scaffold containing the base layer containing high surface areamesoporous silica micro-rods (MSR) and the continuous, fluid supportedlipid bilayer (SLB) but not containing the T-cell activating moleculesand the T-cell co-stimulatory molecules. In one embodiment, inaccordance with the methods of the invention, about a 10-fold to1000-fold, preferably about a 50-fold to 500-fold, or greater, increasein the expansion of the population of T-cells was observed after about 1week of contact with the scaffold compared to a control scaffoldcontaining the base layer containing high surface area mesoporous silicamicro-rods (MSR) and the continuous, fluid supported lipid bilayer (SLB)but not containing the T-cell activating molecules and the T-cellco-stimulatory molecules.

Another aspect of the present invention stems from the surprisingfinding that wherein the method confers increased expansion of thepopulation of T-cells after about 1 week of contact with the scaffold ascompared to a superparamagnetic spherical polymer particle (DYNABEAD)containing the T-cell activating molecules and the T-cell co-stimulatorymolecules. In one embodiment, in accordance with the methods of theinvention, about a 2-fold to 100-fold, preferably about a 5-fold to20-fold, or greater, increase in the expansion of the population ofT-cells was observed after about 1 week of contact with the scaffoldcompared to a superparamagnetic spherical polymer particle (DYNABEAD)containing the T-cell activating molecules and the T-cell co-stimulatorymolecules.

Yet another aspect of the present invention stems from the surprisingfinding that manipulating the T-cells in accordance with theaforementioned methods improves the metabolic activity of T-cells. Inparticular, improved metabolic activity of T-cells was observed after 1week of contact with the scaffold compared to a control scaffoldcontaining the base layer containing high surface area mesoporous silicamicro-rods (MSR) and the continuous, fluid supported lipid bilayer (SLB)but not containing the T-cell activating molecules and the T-cellco-stimulatory molecules. In one embodiment, in accordance with themethods of the invention, about a 2-fold to 100-fold, preferably about a5-fold to 20-fold, or larger, improvement in the metabolic activity ofthe population of T-cells was observed after about 1 week of contactwith the scaffold compared to a control scaffold comprising the baselayer comprising high surface area mesoporous silica micro-rods (MSR)and the continuous, fluid supported lipid bilayer (SLB) but notcontaining the T-cell activating molecules and the T-cell co-stimulatorymolecules.

Another aspect of the present invention stems from the surprisingfinding that the method confers better metabolic activity of thepopulation of T-cells after about 1 week of contact with the scaffoldcompared to a superparamagnetic spherical polymer particle (DYNABEAD)containing the T-cell activating molecules and the T-cell co-stimulatorymolecules. In one embodiment, in accordance with the methods of theinvention, about a 1-fold (e.g., a 100% increase) to 20-fold, preferablya 2-fold to 10-fold increase, or a larger increase, was observed in theexpansion of the population of T-cells was observed after about 1 weekof contact with the scaffold compared to a superparamagnetic sphericalpolymer particle (DYNABEAD) containing the T-cell activating moleculesand the T-cell co-stimulatory molecules.

Additionally, in accordance with the methods of the invention, it wasfound that the expanded T-cells are metabolically active for at leastabout 7 days post-contact with the scaffold. T-cell metabolic activitywas measured via routine techniques, e.g., analyzing levels of cytokineproduction or monitoring cell doublings. Furthermore, in accordance withthe methods of the invention, the expanded T-cells formed larger andmore stable aggregates (e.g., lasting longer) than control scaffolds.For instance, in one experiment, the expanded T-cells formed stableaggregates for at least about 7 days post-contact with the scaffoldwhereas the aggregates had considerably disintegrated in samplesincubated with the control scaffold containing only the MSR base layerand the SLB layer.

Further embodiments of the invention relate to methods for obtaining apolyclonal population of CD8+ cells, comprising, contacting thescaffolds of the invention with a subject's biological sample, therebyactivating, co-stimulating, homeostatically maintaining and optionallyexpanding a population of T-cells present within the sample; contactingthe T-cells in the sample with a reagent for detection of CD8+ cells;and isolating a sub-population of detected CD8+ T-cells from the sample.

In a related embodiment, the instant invention relates to methods forobtaining a polyclonal population of CD4+ cells, comprising, contactingthe scaffolds of the invention with a subject's biological sample,thereby activating, co-stimulating, homeostatically maintaining andoptionally expanding a population of T-cells present within the sample;contacting the T-cells in the sample with a reagent for detection ofCD4+ cells; and isolating a sub-population of detected CD4+ T-cells fromthe sample.

In a related embodiment, the instant invention relates to methods forobtaining a polyclonal population of CD4+/FOXP3+ or CD4+/FOXP3− cells.The method comprises contacting the scaffolds of the invention with asubject's biological sample, thereby activating, co-stimulating,homeostatically maintaining and optionally expanding a population ofT-cells present within the sample; contacting the T-cells in the samplewith a reagent for detection of CD4+ cells; further contacting theT-cells with a reagent for detection of FOXP3+ cells; and isolating asub-population of detected CD4+/FOXP3+ or CD4+/FOXP3− T-cells from thesample. In these embodiments, the reagent for the detection and/orisolation of CD4+ and/or FOXP3+ T-cells is preferably an antibody orantigen-binding fragment thereof which specifically binds to CD4+ andFOXP3 markers. In this context, insofar as FOXP3 is recognized as amaster regulator of the regulatory pathway in the development andfunction of regulatory T cells (which turn the immune response down), itmay be desirable to isolate FOXP3+ cells for certain applications andFOXP3− cells for other applications. For instance, in cancer therapyapplications, it may be desirable to eliminate or reduce regulatory Tcell activity in the T-cell pharmaceutical compositions. Accordingly,the methods may be adapted to screen for FOXP3− cells. Alternately, inthe context of treatment of autoimmune disease, it may be desirable toincrease regulatory T cell activity in the T-cell pharmaceuticalcompositions (as attenuated regulatory T cell activity may becontributing to the body's autoimmune condition). Accordingly, in suchinstances, the formulation methods may be modified to positively screenfor and include FOXP3+ cells.

In yet another embodiment, the instant invention relates to a method forobtaining a polyclonal population of effector memory and/or effectorT-cells. The method comprises contacting the scaffolds of the inventionwith a subject's biological sample, thereby activating, co-stimulating,homeostatically maintaining and optionally expanding a population ofT-cells present within the sample; contacting the T-cells in the samplewith a reagent for detection of CD44+ cells; further contacting theT-cells with a reagent for detection of CD62L; and isolating asub-population of detected CD4+//CD62L+ or CD4+//CD62L− T-cells from thesample. In these embodiments, the effector memory and/or effectorT-cells are preferably CD4+//CD62L−.

In yet another embodiment, the instant invention relates to a method forobtaining a polyclonal population of activated CD8+ T-cells. The methodcomprises contacting the scaffolds of the invention with a subject'sbiological sample, thereby activating, co-stimulating, homeostaticallymaintaining and optionally expanding a population of T-cells presentwithin the sample; contacting the T-cells in the sample with a reagentfor detection of CD8+ cells; further contacting the T-cells with areagent for detection of CD69+; and isolating a sub-population ofdetected CD8+//CD69+ or CD8+//CD69− T-cells from the sample. In theseembodiments, the activated T-cells are preferably CD8+/CD69+.

In yet another embodiment, the instant invention relates to a method forobtaining a polyclonal population of cytotoxin-secreting T-cells. Themethod comprises contacting the scaffolds of the invention with asubject's biological sample, thereby activating, co-stimulating,homeostatically maintaining and optionally expanding a population ofT-cells present within the sample; contacting the T-cells in the samplewith a reagent for detection of CD8+ cells; further contacting theT-cells with a reagent for detection of granzyme B; and isolating asub-population of detected CD8+//granzyme B+ or CD8+//Granzyme B−T-cells from the sample. In these embodiments, the cytotoxin-secretingT-cells are preferably CD8+/Granzyme B+.

In yet another embodiment, the instant invention relates to a method forobtaining a polyclonal population of activator cytokine-secretingT-cells. The method comprises contacting the scaffolds of the inventionwith a subject's biological sample, thereby activating, co-stimulating,homeostatically maintaining and optionally expanding a population ofT-cells present within the sample; contacting the T-cells in the samplewith a reagent for detection of IFNγ+; and isolating a sub-population ofdetected IFNγ+ T-cells from the sample. In these embodiments, theT-cells are preferably IFNγ-secreting cells.

In yet another embodiment, the instant invention relates to a method forobtaining a polyclonal population of memory T-cells. The methodcomprises contacting the scaffolds of the invention with a subject'sbiological sample, thereby activating, co-stimulating, homeostaticallymaintaining and optionally expanding a population of T-cells presentwithin the sample; contacting the T-cells in the sample with a reagentfor detection of CD62L+CCR7+ T-cells; and isolating a sub-population ofdetected CD62L+CCR7+ T-cells from the sample. In these embodiments, theT-cells are preferably CD62L+CCR7+CD4+ central memory T-cells. See,Okada et al., Int Immunol., 20(9):1189-99, 2008. In another embodiment,the instant invention relates to a method for obtaining a polyclonalpopulation of memory T-cells comprising contacting the scaffolds of theinvention with a subject's biological sample, thereby activating,co-stimulating, homeostatically maintaining and optionally expanding apopulation of T-cells present within the sample; contacting the T-cellsin the sample with a reagent for detection of CD62L+CCR7+ T-cells; andisolating a sub-population of detected CD62L-CCR7− T-cells from thesample. In these embodiments, the CD62L-CCR7− T-cells are effectormemory T-cells. See, Sallusto et al., Nature 401: 708-712, 1999.

In yet another embodiment, the instant invention relates to a method fordetecting and/or removing a polyclonal population of exhausted T-cellsfrom a sample. The method comprises contacting the scaffolds of theinvention with a subject's biological sample, thereby activating,co-stimulating, homeostatically maintaining and optionally expanding apopulation of T-cells present in the sample; contacting the T-cells inthe sample with a reagent for detection of CD8+ T cells; furthercontacting the T-cells with a reagent for detection of PD-1+ T-cells;and isolating a sub-population of detected CD8+/PD-1+ T-cells from thesample. The CD8+/PD-1+ T-cells, which indicate exhausted cells, may beoptionally eliminated from the sample.

In another embodiment for detecting and/or removing T-cells from asample, the instant invention provides a method comprising contactingthe scaffolds of the invention with a subject's biological sample,thereby activating, co-stimulating, homeostatically maintaining andoptionally expanding a population of T-cells present within the sample;contacting the T-cells in the sample with a reagent for detection of aco-inhibitory receptor on T-cells; and isolating a sub-population ofT-cells expressing the co-inhibitory receptor from the sample. Theexpression of co-inhibitory receptor generally indicates exhaustedcells, which may be optionally eliminated from the sample. In theseembodiments, the co-inhibitory receptor is a receptor selected from thegroup consisting of CTLA-4, TIM3, LAG3, 2B4, BTLA, CD160, and KLRG1.See, Legat et al., Front Immunol., 2013 Dec. 19; 4:455 In theaforementioned embodiments, the reagents for the detection and/orisolation of cells are preferably an antibodies or antigen-bindingfragments thereof, e.g., antibodies which specifically bind to theaforementioned markers, e.g., CD8, CD4, FOXP3, CD62L, PD-1, granzyme B,etc. The detection of these cell-surface markers is preferably carriedout using FACS analysis.

The invention further relates to isolating polyclonal T-cell populationsusing one or more of the aforementioned methods and further detectingthe production of a cytokine selected from the group consisting ofinterferon gamma (IFNγ), tissue necrosis factor alpha (TNFα), IL-2,IL-1, IL-4, IL-5, IL-10, and IL-13, or a combination thereof. Thecytokines may permit validation of the isolation methods. For instance,wherein the manipulated T-cells are T-helper 1 (Th1) cells, the methodsmay comprise detecting the production of a cytokine selected from thegroup consisting of IL-2, interferon gamma (IFNγ) and tissue necrosisfactor alpha (TNFα), or a combination thereof. Likewise, wherein themanipulated T-cells are T-helper 2 (Th2) cells and the method comprisesdetecting the production of a cytokine selected from the groupconsisting of IL-4, IL-5, IL-10 and IL-13, or a combination thereof.Furthermore, wherein the manipulated T-cells are cytotoxic T (Tc) cells,the methods may further comprise detecting the production of a cytokineselected from the group consisting of interferon gamma (IFNγ) andlymphotoxin alpha (LTα/TNFβ), or a combination thereof optionallytogether with the detection of a secreted cytotoxin selected from thegroup consisting of a granzyme or a perforin, or a combination thereof.

Using certain methodologies it may be advantageous to maintain long-termstimulation of a population of T cells following the initial activationand stimulation, by separating the T cells from the stimulus after aperiod of about 12 to about 14 days. The rate of T cell proliferation ismonitored periodically (e.g., daily) by, for example, examining the sizeor measuring the volume of the T cells, such as with a Coulter Counter.In this regard, a resting T cell has a mean diameter of about 6.8microns, and upon initial activation and stimulation, in the presence ofthe stimulating ligand, the T cell mean diameter will increase to over12 microns by day 4 and begin to decrease by about day 6. When the meanT cell diameter decreases to approximately 8 microns, the T cells may bereactivated and re-stimulated to induce further proliferation of the Tcells. Alternatively, the rate of T cell proliferation and time for Tcell re-stimulation can be monitored by assaying for the presence ofcell surface molecules, such as, a cell surface marker selected from thegroup consisting of CD69, CD4, CD8, CD25, CD62L, FOXP3, HLA-DR, CD28,and CD134, or a combination thereof. Additionally, the methods may becomplemented by assaying for the presence of non T-cell surfacemolecules, such as, CD36, CD40, and CD44, or a combination thereof. Incertain instances, the methods may be complemented by assaying for thepresence of non T-cell surface molecules, such as, CD154, CD54, CD25,CD137, CD134, which are induced on activated T cells.

Diagnosis and Therapy of Diseases

Embodiments described herein further relate to methods for treating adisease or a disorder in a subject. In one embodiment, the disease iscancer. In another embodiment, the disease is an autoimmune disorder. Ina third embodiment, the disease is a disease caused by a pathogen.

In these embodiments, a subject with a disease may be treated bycontacting the subject's sample comprising a T-cell population with theantigen presenting cell-mimetic scaffold (APC-MS) of the invention,thereby activating, co-stimulating and homeostatically maintaining thepopulation of T-cells; optionally expanding the population of T-cells;and administering the activated, co-stimulated, maintained andoptionally expanded T-cells into the subject, thereby treating thedisease in the subject. In one embodiment, the T-cell population iscontacted with the scaffold for a period, e.g., 0.5 day, 1 day, 2 days,3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11days, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days, 18 days, 19days, 20 days, 21 days, 25 days, 30 days, 35 days, 38 days, 45 days, 50days, 60 days, or more, and the cells contained therein are manipulatedusing one or more of the aforementioned techniques. Examples ofmanipulation include, for example, activation, division,differentiation, growth, expansion, reprogramming, anergy, quiescence,senescence, apoptosis, death, etc. The cells need not be physicallyremoved from the scaffold to be manipulated. Thus in one embodiment, thescaffolds are contacted with the subject's sample in situ (e.g., byimplanting the scaffold into the subject). In other embodiments, thecells are manipulated ex situ (e.g., incubating the scaffold and thesubject's withdrawn blood sample).

In the therapeutic embodiments of the invention, the T cellsadministered to the mammal are about 4 to about 35 days old, whereuponthe regression of the disease in the mammal is promoted. In someembodiments, the administered T cells are less than about 14 days old,e.g., about 7 to about 21 days old. The inventive methods providenumerous advantages. For example, T cells that are about 4 to about 14days old are believed to provide improved in vivo proliferation,survival, and activity as compared to T cells that are about 60 days oldor older. The period of time required to generate T cells for adoptivecell therapy (ACT) may be shortened from an average of about 44 days toa range of about 4 to about 15 days (or less than about 35 days, e.g.,about 7 to about 15 days). Accordingly, more patients may be treatedbefore their disease burden progresses to a stage in whichadministration of ACT may no longer be safe or possible. Furthermore,because preferred embodiments of the inventive methods do not require invitro testing of specific antigen reactivity prior to administration,the inventive methods reduce the time, expense, and labor associatedwith the treatment of patients. Additionally, the inventive methods mayadvantageously administer T cells that are pooled from bulk culturesinstead of those derived from microcultures. The development of asimpler and faster method to generate clinically effective T cells isbelieved to aid in the more widespread use of adoptive cell therapy. Theinventive methods also advantageously utilize T cell cultures that couldbe falsely predicted to be unreactive in vivo by in vitro testing ofspecific antigen reactivity. Because T cell cultures generated from asingle tumor specimen have diverse specific reactivities, the lack of invitro antigen reactivity testing advantageously avoids having to chooseonly a few T cell cultures to expand, and therefore provides a morediverse repertoire of tumor reactivities to be administered to thepatient. T cells that are about 4 to about 30 days old also contain agreater diversity of cells and a higher frequency of active/healthycells than T cells. In addition, one or more aspects (e.g., but notlimited to, culturing and/or expanding) of the inventive methods may beautomatable.

An embodiment of the method comprises culturing autologous T cells.Tumor samples are obtained from patients and a single cell suspension isobtained. The single cell suspension can be obtained in any suitablemanner, e.g., mechanically (disaggregating the tumor using, e.g., aGENTLEMACS™ Dissociator, Miltenyi Biotec, Auburn, Calif.) orenzymatically (e.g., collagenase or DNase). Single-cell suspensions oftumor enzymatic digests are cultured in scaffolds or scaffolds of theinvention. The cells are cultured until confluence (e.g., about 2×10⁶lymphocytes), e.g., from about 2 to about 21 days, preferably from about4 to about 14 days. For example, the cells may be cultured from 5 days,5.5 days, or 5.8 days, 6.0 days, 6.5 days, 7.0 days to 21 days, 21.5days, or 21.8 days, preferably from 10 days, 10.5 days, or 10.8 days to14 days, 14.5 days, or 14.8 days.

An embodiment of the method comprises expanding cultured T cells. Thecultured T cells are pooled and rapidly expanded. Rapid expansionprovides an increase in the number of antigen-specific T-cells of atleast about 10-fold (e.g., 10-, 20-, 40-, 50-, 60-, 70-, 80-, 90-, or100-fold, or greater) over a period of about 7 to about 14 days,preferably about 14 days. More preferably, rapid expansion provides anincrease of at least about 200-fold (e.g., 200-, 300-, 400-, 500-, 600-,700-, 800-, 900-, or greater) over a period of about 7 to about 14 days,preferably about 14 days. Most preferably, rapid expansion provides anincrease of at least about 400-fold or greater over a period of about 10to about 14 days, preferably about 14 days. Optionally, the cells mayundergo initial expansion in the scaffolds, upon which they are subjectto rapid expansion. Under this two-step expansion protocol, an increaseof about 1000-fold over a period of about 7 to 14 days may be achieved.

Expansion can be accomplished by any of a number of methods as are knownin the art. For example, T cells can be rapidly expanded usingnon-specific T-cell receptor stimulation in the presence of feederlymphocytes and either interleukin-2 (IL-2) or interleukin-15 (IL-15),with IL-2 being preferred. The non-specific T-cell receptor stimulus caninclude around 30 ng/ml of OKT3, a mouse monoclonal anti-CD3 antibody(available from Ortho-McNeil®, Raritan, N.J.). Alternatively, T cellscan be rapidly expanded by stimulation of peripheral blood mononuclearcells (PBMC) in vitro with one or more antigens (including antigenicportions thereof, such as epitope(s), or a cell) of the cancer, whichcan be optionally expressed from a vector, such as an human leukocyteantigen A2 (HLA-A2) binding peptide, e.g., 0.3 μM MART-1:26-35 (27 L) orgp100:209-217 (210M), in the presence of a T-cell growth factor, such as300 IU/ml IL-2 or IL-15, with IL-2 being preferred. The in vitro-inducedT-cells are rapidly expanded by re-stimulation with the same antigen(s)of the cancer pulsed onto HLA-A2-expressing antigen-presenting cells.Alternatively, the T-cells can be re-stimulated with irradiated,autologous lymphocytes or with irradiated HLA-A2+ allogeneic lymphocytesand IL-2, for example.

An embodiment of the method comprises administering to the subject, theexpanded T cells, wherein the T cells administered to the mammal areabout 4 to about 35 days old. For example, the administered cells may be6, 7, or 8 to 14, 15, or 16 days old. In some embodiments, the T cellsadministered to the mammal are about 4 to about 29 or about 7 to about15 days old, or about 10 days old. In this regard, the T cells that areadministered to the mammal according to an embodiment of the inventionare “young” T cells, i.e., minimally cultured T cells.

Young T cell cultures that are administered to the mammal in accordancewith an embodiment of the invention advantageously have featuresassociated with in vivo persistence, proliferation, and antitumoractivity. For example, young T cell cultures have a higher expression ofCD27 and/or CD28 than T cells that are about 44 days old. Without beingbound to a particular theory, it is believed that CD27 and CD28 areassociated with proliferation, in vivo persistence, and a lessdifferentiated state of T cells (the increased differentiation of Tcells is believed to negatively affect the capacity of T cells tofunction in vivo). T cells expressing higher levels of CD27 are believedto have better antitumor activity than CD27-low cells. Moreover, young Tcell cultures have a higher frequency of CD4+ cells than T cells thatare about 44 days old.

In addition, young T cell cultures have a mean telomere length that islonger than that of T cells that are about 44 days old. Without beingbound to a particular theory, it is believed that T cells lose anestimated telomere length of 0.8 kb per week in culture, and that youngT cell cultures have telomeres that are about 1.4 kb longer than T cellsthat are about 44 days old. Without being bound to a particular theory,it is believed that longer telomere lengths are associated with positiveobjective clinical responses in patients, and persistence of the cellsin vivo.

The T-cells can be administered by any suitable route as known in theart. Preferably, the T-cells are administered as an intra-arterial orintravenous infusion, which preferably lasts about 30 to about 60minutes. Other examples of routes of administration includesubcutaneous, intraperitoneal, intrathecal and intralymphatic.

Additionally, embodiments of the instant invention provide for variousmodes of administering the therapeutic compositions comprising theexpanded cells. In one embodiment, the expanded cells are first purifiedand then administered into a subject. Alternately, the expanded cellsmay be mixed with the scaffolds of the invention prior to administrationinto the subject. Under this alternate approach, the scaffolds (APC-MS)may continue to stimulate cells in vivo and may also function toselectively manipulate target whole blood cells in the in vivo setting.

The therapeutic methods of the invention may involve re-stimulating thepopulation of T-cells prior to the administration step. There-stimulation step may be carried out using art-known techniques. Inone embodiment, the re-stimulation step is carried out by re-incubatingthe cells with the scaffold composition. In another embodiment,re-stimulation is carried out by addition of phorbol 12-myristate13-acetate (PMA, 10 ng/ml, Sigma-Aldrich, Inc.), ionomycin (0.5 μg/ml,Sigma-Aldrich, Inc.) and Brefeldin A (eBiosciences, Inc.). In yetanother embodiment, the re-stimulation step is carried out by includingan antigen (e.g., a pathogenic antigen or a cancer antigen) in thescaffold or extrinsically in the culture.

In one embodiment, the therapeutic methods are conducted by manipulatingT-cells that are obtained from a blood sample, a bone marrow sample, alymphatic sample or a splenic sample of a subject.

Accordingly, embodiments of the instant invention provide for methodsfor treating cancer in a subject. The method comprises contacting thesubject's sample comprising a T-cell population with the antigenpresenting cell-mimetic scaffold (APC-MS) of the invention, therebyactivating, co-stimulating and homeostatically maintaining thepopulation of T-cells; optionally expanding the population of T-cells;and administering the activated, co-stimulated, maintained andoptionally expanded T-cells into the subject, thereby treating thecancer in the subject. In certain embodiments, the scaffolds may beprovided with a cancer antigen. In one embodiment, the cancer antigen ispresented, e.g., for recognition by T-cells, in an MHC molecule or afragment thereof. In certain instances, whole cell products may beprovided.

Representative examples of cancer antigens include, but are not limitedto, MAGE-1, MAGE-2, MAGE-3, CEA, Tyrosinase, midkin, BAGE, CASP-8,β-catenin, β-catenin, γ-catenin, CA-125, CDK-1, CDK4, ESO-1, gp75,gp100, MART-1, MUC-1, MUM-1, p53, PAP, PSA, PSMA, ras, trp-1, HER-2,TRP-1, TRP-2, IL13Ralpha, IL13Ralpha2, AIM-2, AIM-3, NY-ESO-1, C9orf112, SART1, SART2, SART3, BRAP, RTN4, GLEA2, TNKS2, KIAA0376, ING4,HSPH1, C13orf24, RBPSUH, C6orf153, NKTR, NSEP1, U2AF1L, CYNL2, TPR,SOX2, GOLGA, BMI1, COX-2, EGFRvIII, EZH2, LICAM, Livin, Livinβ, MRP-3,Nestin, OLIG2, ART1, ART4, B-cyclin, Gli1, Cav-1, cathepsin B, CD74,E-cadherin, EphA2/Eck, Fra-1/Fosl 1, GAGE-1, Ganglioside/GD2, GnT-V,β1,6-N, Ki67, Ku70/80, PROX1, PSCA, SOX10, SOX11, Survivin, UPAR, WT-1,Dipeptidyl peptidase IV (DPPIV), adenosine deaminase-binding protein (ADAbp), cyclophilin b, Colorectal associated antigen (CRC)-C017-1A/GA733,T-cell receptor/CD3-zeta chain, GAGE-family of tumor antigens, RAGE,LAGE-I, NAG, GnT-V, RCAS1, α-fetoprotein, pl20ctn, Pmel117, PRAME, brainglycogen phosphorylase, SSX-I, SSX-2 (HOM-MEL-40), SSX-I, SSX-4, SSX-5,SCP-I, CT-7, cdc27, adenomatous polyposis coli protein (APC), fodrin,PlA, Connexin 37, Ig-idiotype, p15, GM2, GD2 gangliosides, Smad familyof tumor antigens, Imp-1, EBV-encoded nuclear antigen (EBNA)-I,UL16-binding protein-like transcript 1 (Mult1), RAE-1 proteins, H60,MICA, MICB, and c-erbB-2, or an immunogenic peptide thereof, andcombinations thereof.

In another embodiment, the cancer antigen is a neoantigen identified ina patient. A neoantigenic determinant is an epitope on a neoantigen,which is a newly formed antigen that has not been previously recognizedby the immune system. Neoantigens are often associated with tumorantigens and are found in oncogenic cells. Neoantigens and, byextension, neoantigenic determinants can be formed when a proteinundergoes further modification within a biochemical pathway such asglycosylation, phosphorylation or proteolysis, leading to the generationof new epitopes. These epitopes can be recognized by separate, specificantibodies. See, Schumacher et al., Science 348 (6230): 69-74, 2015. Inone embodiment, the neoantigen may be detected in a patient-specificmanner. Methods for detecting neoantigens from a patient sample, e.g.,blood sample, are described in U.S. Pat. No. 9,115,402, which isincorporated by reference herein, In one embodiment, the neoantigen is apeptide derived from SF3B1, MYD88, TP53, ATM, Abl, A FBXW7, a DDX3X,MAPK1, GNB1, CDK4, MUM1, CTNNBI, CDC27, TRAPPCI, TPI, ASCC3, HHAT, FN1,OS-9, PTPRK, CDKN2A, HLA-AlI, GAS7, GAPDH, SIRT2, GPNMB, SNRP116,RBAF600, SNRPDI, Prdx5, CLPP, PPPIR3B, EF2, ACTN4, ME1, NF-YC, HLA-A2,HSP70-2, KIAA1440, CASP8, or a combination thereof. See, Lu et al.,Seminars in Immunology, 28(1), 22-27, 2016.

In practicing the cancer therapeutic embodiments outlined above, it maybe advantageous to provide scaffolds that have been fabricated withcytotoxic T-cell-specific activating molecules and cytotoxicT-cell-specific co-stimulatory molecules, optionally together with oneor more additional agents that confer activation, division,differentiation, growth, expansion, or reprogramming of cytotoxic Tcells. Representative examples of such molecules and agents have beenprovided above.

In certain embodiments, the sequestered and/or isolated cells may begenetically modified. In one embodiment, the effector cells aregenetically modified to express a chimeric antigen receptor (CAR)specific for CD19 (CD19 CAR-T cells). This particular type of T-cellshas produced a high rate of complete remission (CR) in adult andpediatric patients with relapsed and refractory B cell acutelymphoblastic leukemia (B-ALL) in small phase I clinical trials. See,Turtle et al. (J Clin Invest., 126, 2123-38, 2016) and the referencescited therein. Favorable results have also been seen in clinical trialsof CD19 CAR-T cell therapy in non-Hodgkin's lymphoma (NHL) and chroniclymphocytic leukemia (CLL). These studies suggest that robustproliferation of transferred CAR-T cells in the recipient correlateswith clinical response and that prolonged in vivo persistence offunctional CAR-T cells may prevent disease relapse. Accordingly, in oneembodiment, the invention relates to methods for further formulatingT-cell compositions for cancer therapy, comprising, further geneticallymodifying the T-cells obtained from the scaffolds. The geneticmodification may be mediated ex situ or in situ. Any technique may beused to genetically modify T-cells, including, but not relating to,using viral vectors, plasmids, transposon/transposase systems, shRNA,siRNA, antisense RNA, and the like. In some embodiments, the T-cell hasbeen genetically-modified using a gene editing system (e.g., aCRISPR/Cas9 system). In some embodiments, the isolated T-cells aregenetically modified using a viral delivery system. In some embodiments,the isolated T-cells are genetically modified using a lentiviral system.In some embodiments, the isolated T-cells are genetically modified usinga retroviral system. In some embodiments, the isolated T-cells aregenetically modified using an adenoviral system. In some embodiments,the isolated T-cells are contacted with an agent that promotesinteraction with the viral delivery system or viral sequestration (e.g.,an agent that promotes receptor-mediated interactions with the viraldelivery system or agents that promote electrostatic interactions withthe viral delivery system).

In some embodiments, the isolated T-cells are genetically modified usinga viral delivery system in situ. In embodiments where the isolatedT-cells are genetically modified in situ the scaffold may comprise anagent that promotes viral sequestration. The agent(s) that promote viralsequestration may be present on the surface of the lipid bilayer of theMSR-SLB either through adsorption or by attachment to a lipid headgroup.In some embodiments, the agent that promotes viral sequestration is afibronectin peptide, such as RetroNectin®. In some embodiments, theagent that promotes viral sequestration is an amphipathic peptide, suchas Vectofusin-1®. In some embodiments, the scaffold may further comprisea T-cell activating molecule, a T-cell co-stimulatory molecule and/or aT-cell homeostatic agent. Without wishing to be bound by any particulartheory, it is believed that when a T-cell is contacted with a scaffoldcomprising an agent that promotes sequestration in combination with aT-cell activating molecule, a T-cell co-stimulatory molecule and/or aT-cell homeostatic agent, the scaffold may facilitate the activation andexpansion of T-cells which may lead to cell clustering and allow for aviral delivery system to be in close proximity with the T-cells therebypromoting more efficient transduction of the cells. The T-cellactivating molecule, a T-cell co-stimulatory molecule and/or a T-cellhomeostatic agent present on the scaffold may be selected to result inthe desired T-cell phenotype which may enhance the therapeutic efficacyof the resulting T-cell (see, e.g., Sommermeyer et al, Leukemia 30(2):492-500 (2016)).

In some embodiments, the isolated T-cells are genetically modified toexpress a chimeric antigen receptor (CAR). In one embodiment, CD4+ andCD8+ T cells are lentivirally transduced to express the CD19 CAR and atruncated human epidermal growth factor receptor (EGFRt) that enablesidentification of transduced cells by flow cytometry using the anti-EGFRmonoclonal antibody cetuximab. Transduced EGFRt+ CD4+ and CD8+ T cellsare enriched during culture by a single stimulation with irradiatedCD19+ lymphoblastoid cell line (LCL). The median frequency ofEGFRt+CAR-T cells within the CD3+CD4+ and CD3+CD8+ subsets in theproducts at release for infusion, which confers good therapeuticoutcome, is about 80% (range 50.0%-95.9%) and about 85% (range13.0%-95.6%), respectively. See, Turtle et al. (J Clin Invest., 126,2123-38, 2016). The genetically modified T-cells may be further expandedby incubating the T-cell product with the scaffolds of the invention. Inone embodiment, scaffolds containing CAR T-cell-specific antigens, e.g.,CD19, CD22 or a fragment thereof or a variant thereof, may be employedto selectively expand the desired CAR T-cells.

In certain embodiments, the scaffolds are provided with products thatare useful in practicing the cancer therapy methods. Representativeexamples include, for example, hybridomas of B-cells, stable lineages ofT-cells, antibodies derived from B-cells or hybridomas thereof,receptors which bind to the cancer antigens (receptors which bind to MHCmolecules presenting the antigens), including fragments thereof, nucleicacids encoding the receptors or antigen-binding domains thereof, nucleicacids encoding antibodies, including whole cells.

Embodiments of the instant invention provide for methods for treating animmunodeficiency disorder in a subject comprising contacting thesubject's sample comprising a T-cell population with the antigenpresenting cell-mimetic scaffold (APC-MS) of the invention, therebyactivating, co-stimulating and homeostatically maintaining thepopulation of T-cells; optionally expanding the population of T-cells;and administering the activated, co-stimulated, maintained andoptionally expanded T-cells into the subject, thereby treating theimmunodeficiency disorder in the subject.

In one embodiment, there is provided a method for treating animmunodeficiency disorder selected from the group consisting of primaryimmunodeficiency disorder and acquired immunodeficiency disorder,comprising contacting the subject's sample comprising a T-cellpopulation with the APC-MS of the invention, thereby activating,co-stimulating and homeostatically maintaining the population ofT-cells; optionally expanding the population of T-cells; andadministering the activated, co-stimulated, maintained and optionallyexpanded T-cells into the subject, thereby treating the immunodeficiencydisorder in the subject. In one embodiment, the immunodeficiencydisorder may be an acquired immunodeficiency disorder, e.g., acquiredimmunodeficiency syndrome (AIDS) or a hereditary disorder, e.g.,DiGeorge syndrome (DGS), chromosomal breakage syndrome (CBS), ataxiatelangiectasia (AT) and Wiskott-Aldrich syndrome (WAS), or a combinationthereof.

In practicing the therapy of immunodeficiency disorders, as outlinedabove, it may be advantageous to provide scaffolds that have beenfabricated with helper T-cell-specific activating molecules and helperT-cell-specific co-stimulatory molecules, optionally together with oneor more additional agents that confer activation, division,differentiation, growth, expansion, or reprogramming of helper T cells.Representative examples of such molecules and agents have been providedabove.

Embodiments of the instant invention provide for methods for treating apathogenic disease in a subject comprising contacting the subject'ssample comprising a T-cell population with the antigen presentingcell-mimetic scaffold (APC-MS) of the invention, thereby activating,co-stimulating and homeostatically maintaining the population ofT-cells; optionally expanding the population of T-cells; andadministering the activated, co-stimulated, maintained and optionallyexpanded T-cells into the subject, thereby treating the pathogenicdisease in the subject. In some instances, the immune cells orcompositions derived from the manipulation step may be administeredprophylactically, e.g., before the onset of the disease symptoms in thesubject. Pathogenic diseases that may be treated in accordance with theaforementioned embodiment include, bacterial diseases, viral diseases,fungal diseases, or a combination thereof.

Embodiments of the instant invention provide for methods for treating anautoimmune disease in a subject. The method comprises contacting thesubject's sample comprising a T-cell population with the antigenpresenting cell-mimetic scaffold (APC-MS) of the invention, therebyactivating, co-stimulating and homeostatically maintaining thepopulation of T-cells; optionally expanding the population of T-cells;and administering the activated, co-stimulated, maintained andoptionally expanded T-cells into the subject, thereby treating theautoimmune disease in the subject.

In the context of treating autoimmune diseases, it may be preferable notto administer active immune cells (as these are autoreactive) but ratherquiescent, senescent or inactivated immune cells. Preferably, the immunecells are T-cells. Alternately, regulators of immune cells e.g.,regulatory T cells or suppressor T cells, may be administered. Thescaffolds/devices may be fabricated for the manipulation of Ts/Treg cellsub-populations, which, are then administered into subjects.

Accordingly, in some embodiments, the invention provides for a methodfor treating an autoimmune disease by administering to subject in needthereof, the scaffold of the invention, wherein the plurality ofantigens in the scaffold are specific for the autoimmune disease,collecting a plurality of regulatory or suppressor T-cells in thescaffold/device, wherein the plurality of regulatory or suppressorT-cells are specific to the autoimmune antigens, and administering theplurality of regulatory T-cells or suppressor T-cells or productsderived therefrom into the subject, thereby treating the autoimmunedisease.

Cell products that are useful in practicing the therapy of autoimmunediseases include, for example, antibodies and receptors which bind toautoreactive cells, regulatory proteins located in suppressor orregulatory T-cells, including nucleic acid sequences which encode suchmolecules.

In the therapeutic embodiments described above, cells may be formulatedat total cell concentrations including from about 5×10² cells/ml toabout 1×10⁹ cells/ml. Preferred doses of T cells range from about 2×10⁶cells to about 9×10⁷ cells.

Embodiments of the instant invention further relate to therapy ofdiseases by administering one or more of the aforementionedcompositions. The composition may be a pharmaceutical composition, whichis administered by any means that achieve their intended purpose. Forexample, administration may be by parenteral, subcutaneous, intravenous,intraarterial, intradermal, intramuscular, intraperitoneal, transdermal,transmucosal, intracerebral, intrathecal, or intraventricular routes.Alternatively, or concurrently, administration may be by the oral route.The pharmaceutical compositions may be administered parenterally bybolus injection or by gradual perfusion over time.

The dosage administered may be dependent upon the age, sex, health, andweight of the recipient, kind of concurrent treatment, if any, frequencyof treatment, and the nature of the effect desired. The dose ranges forthe administration of the pharmaceutical compositions may be largeenough to produce the desired effect, whereby, for example, autoreactiveT cells are depleted and/or the autoimmune disease is significantlyprevented, suppressed, or treated. The doses may not be so large as tocause adverse side effects, such as unwanted cross reactions,generalized immunosuppression, anaphylactic reactions and the like.

Embodiments described herein further relate to methods for detecting ordiagnosing a disease or a disorder in a subject. Any disease or disordermay be detected or diagnosed using the aforementioned methods.Particularly preferably, the disease is an autoimmune disease selectedfrom the group consisting of rheumatoid arthritis, lupus, celiacdisease, inflammatory bowel disease or Crohn's disease, sjögren'ssyndrome polymyalgia rheumatic, multiple sclerosis, ankylosingspondylitis, Type 1 diabetes, alopecia areata, vasculitis, temporalarteritis, etc. In other embodiments, the disease is a cancer which isselected from the group consisting of head and neck cancer, breastcancer, pancreatic cancer, prostate cancer, renal cancer, esophagealcancer, bone cancer, testicular cancer, cervical cancer,gastrointestinal cancer, glioblastoma, leukemia, lymphoma, mantle celllymphoma, pre-neoplastic lesions in the lung, colon cancer, melanoma,and bladder cancer. Pathogenic diseases that may be diagnosed inaccordance with the aforementioned embodiment include, bacterialdiseases, viral diseases, fungal diseases, or a combination thereof.

In these embodiments, a subject with a disease may be diagnosed by firstcontacting a subject's sample containing the immune cell of interestwith a scaffold of the invention, wherein the antigens in the scaffoldare specific to the disease. In one embodiment, the sample containsT-cells and the scaffold/device is contacted with the sample for aperiod, e.g., 0.5 days, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15days, 16 days, 17 days, 18 days, 19 days, 20 days, 21 days, 25 days, 30days, 35 days, 40 days, 45 days, 50 days, 60 days, or more, and thecells in the scaffold are analyzed using one or more of theaforementioned techniques. For example, in the context of diagnosingautoimmune diseases, the cells that are analyzed may include activatedT-cells. In the context of cancer diagnosis, the cells that are analyzedmy include tumor-antigen specific T-cells. In the context of pathogenicdiseases, the cells that are analyzed may include T-cells whichspecifically eliminate the pathogens (e.g., by analyzing Th1 cells incase of intracellular pathogens and Th2 cells in case of extracellularpathogens).

The subject is an animal, preferably a mammal or a bird. Particularlypreferably, the subject is selected from the group consisting of humans,dogs, cats, pigs, cows, buffalo and horses. Most preferably, the subjectis a human. Any immune cell may be used in the diagnosis of the diseaseor disorder. Preferably, diagnosis is performed with a lymphocyte, e.g.,T-cells.

The analytical step may be carried out using any routine methods.Accordingly, in one embodiment, the analytical step may involvedetermining the number of immune cells that are specific to theautoimmune disease. Any routine technique may be used to determineantigen-binding specificity of immune cells, e.g., loading cell samplesonto antigen-coated surfaces, washing away non-specifically bound cells,and quantitating the number of antigen-specific cells (either in freeform by releasing the bound cells or in bound form) using a detectionagent (e.g., an antibody that binds to a cell-surface epitope located onthe antigen-specific cells). In another embodiment, the analytical stepmay involve determining the physical or biological characteristics ofthe antigen-specific immune cells. Examples of physical characteristicsinclude, for example, size, shape, reflectivity, morphology, density.Examples of biological characteristics include, for example, expressionof particular cell surface markers, secretion of cytokines, reactivityto particular antigens or agents, patterns of gene expression.

The analytical step may be tied to a correlation step, wherein, theresults of the analytical step are correlated to the parameter ofinterest. Representative types of parameters include, presence (orabsence of disease), type of disease (e.g., aggressive vs.non-aggressive autoimmune disorder; druggable vs. non-druggable disease,e.g., antibiotic susceptible vs. antibiotic resistant bacterialinfection, immunotherapy-resistant vs. immunotherapy-sensitive cancer),stage of disease, progression/regression of disease (over time), etc. Inone embodiment, the parameter relates to presence or absence of disease(which can be expressed in binary terms). In another embodiment, theparameter relates to staging of disease (which can be expressed in anominal scale, e.g., stage I-IV, with stage IV being the highest). Yetin another embodiment, the parameter relates to odds or likelihood ofoccurrence of the disease, e.g., at least 20%, 30%, 40%, 50%, 60%, 70%,80%, 2-fold, 3-fold, 5-fold, 10-fold, 20-fold or more.

In the aforementioned diagnostic methods the parameters may be comparedto a baseline value. The baseline value may be a value that ispre-determined, e.g., in a population of healthy subjects. For example,where the antigen of interest is rheumatoid arthritis (RA) antigen, abaseline level of RA-specific antibodies (or T-cells) in healthysubjects may be used in the correlation step. Alternately, the baselinevalue may be experimentally identified using suitable controls. Theskilled worker can use routine techniques to correlate and/or drawinferences between various subject groups.

Accordingly, embodiments of the invention relate to detecting ordiagnosing autoimmune disease, cancer, or a pathogenic disease in asubject by contacting a subject's sample with the scaffolds of theinvention containing antigens which are specific to the autoimmunedisease, cancer disease, or pathogenic disease, and analyzing the immunecells contained therein. The contacting step may be performed in vivo(e.g., by implanting the scaffold in a subject) or ex vivo (e.g., byculturing a blood sample withdrawn from a subject with the scaffold). Incertain embodiments, the analytical step may be performed by firstremoving the immune cells from the scaffolds using routine techniques,i.e., via ex situ analysis. For instance, mild detergents and enzymesmay be used to dislodge the cells from the scaffolds. Alternately, thedetection/analytical steps may be carried out without removing the cellsfrom the scaffolds, i.e., via in situ analysis.

Related embodiments are directed to methods of monitoring theprogression of a disease in a subject. The method comprises contacting asubject's sample with the scaffolds of the invention containing antigensthat are specific to the disease and analyzing the immune cellscontained therein. The number/types of immune cells contained in thedevice may offer valuable cues as to the progression of the disease.Alternately, wherein the subject has undergone therapeutic intervention,analogous methods may be used to monitor the therapy of disease and/ordisease management.

The aforementioned methods may be used to monitor theprogression/therapy of autoimmune disorders, cancers, pathogenicdiseases, and the like. Preferably, the immune cells that are used inthe diagnostic methods are T-cells.

In the context of autoimmune disorders, the progression of the diseasemay be monitored by analyzing the number and/or type of autoreactive Tcells. Depending on the result of the analysis, methods of interventionand/therapy may be designed to minimize the severity of the symptoms. Inother instances, preventive methods may be undertaken, includingproviding recommendations to subjects on dietary, nutritional and/orother lifestyle changes.

Embodiments described herein further relate to methods for devising andproducing novel compositions for treating a disease. The methodcomprises administering the scaffolds of the invention containingdisease specific antigens to a subject, which then manipulate immunecells that are specific to the disease, optionally isolating, enriching,and expanding the immune cells manipulated in the device, and thenadministering the immune cells back to the subject. Alternately,products derived from such immune cells may be administered to thesubjects. Examples of products derived from the immune cells include,nucleic acids (including vectors and cells containing such nucleicacids), peptides, proteins, antibodies, cytokines, etc.

Preferably, the disease is an autoimmune disease. In one embodiment,autoreactive T cells which have been isolated (and optionally expandedin culture as described herein) by the aforementioned methods may beinactivated in situ or ex situ. Methods of inactivating T cells areknown in the art. Examples include, but not limited to, chemicalinactivation or irradiation. The autoreactive T cells may be preservedeither before or after inactivation using a number of techniques knownto those skilled in the art including, but not limited to,cryopreservation. As described below, the composition may be used as avaccine to deplete autoreactive T cells in autoimmune patients.

Embodiments described herein further relate to compositions and vaccinesproduced by the aforementioned methods. The composition may be apharmaceutical composition, which may be produced using methods wellknown in the art. Pharmaceutical compositions used as preclinical andclinical therapeutics in the treatment of disease or disorders may beproduced by those of skill, employing accepted principles of diagnosisand treatment.

In one embodiment, the vaccine may comprise autoreactive T cellscomprising homogeneous (“monoclonal”) or heterogeneous (“polyclonal”)patterns of Vβ-Dβ-Jβgene usage. Clinical studies indicate thatautoimmune patients receiving autologous monoclonal T cell vaccinationmay show a gradual decline in the immunity against autoreactive T cells.In some cases, the reappearing autoreactive T cells may originate fromdifferent clonal populations, suggesting that the T cells may undergoclonal shift or epitope spreading potentially associated with theongoing disease process. Clonal shift or epitope spreading may be aproblem in autoimmune diseases mediated by autoreactive T cells. Avaccine comprising polyclonal autoreactive T cells capable of depletingmultiple populations of autoreactive T cells may avoid problems withclonal shift or epitope spreading. The compositions/vaccines of theinvention containing desired T-cells may be provided with apharmaceutically acceptable carrier. Lyophilized preparations of T-cellsmay be provided as well.

IV. Kits/Devices

In certain embodiments, the present invention provides kits comprising,in one or separate compartments, the scaffolds of the instant invention.The kits may further comprise additional ingredients. The kits mayoptionally comprise instructions for formulating the scaffolds fordiagnostic or therapeutic applications. The kits may also compriseinstructions for using the kit components, either individually ortogether, in the therapy or diagnosis of various disorders and/ordiseases.

In a related embodiment, the present invention provides kits comprisingthe scaffolds of the invention along with reagents for selecting,culturing, expanding, sustaining, and/or transplanting the manipulatedcells of interest. Representative examples of cell selection kits,culture kits, expansion kits, transplantation kits for T-cells, B-cellsand antigen presenting cells are known in the art.

This invention is further illustrated by the following examples whichshould not be construed as limiting. The entire contents of allreferences, patents and published patent applications cited throughoutthis application, as well as the Figures are hereby incorporated hereinby reference.

EXAMPLES Example 1: Construction of Scaffolds and Microscopic Analysisof the Assembled Structures

Antigen-presenting cells-mimetic scaffolds (APC-MS) were assembled usingthe methodology described below. Briefly, a base layer containing highsurface area mesoporous silica micro-rods (MSR) was first provided, ontowhich various T-cell homeostatic agents, e.g., interleukins such as IL2and/or cytokines such as TGF-beta, are optionally loaded. In certainembodiments, it may be preferable to load the homeostatic agents on tothe MSR layer. Then, a continuous, fluid supported lipid bilayer (SLB)was layered on the base layer, thereby generating an MSR-SLB scaffold.If the homeostatic agents are not directly loaded on the MSR layer, thenthey can be loaded after SLB payload has been applied on top of the MSRlayer. Then, a blocking agent such as BSA may be applied to blocknon-specific integration sites in the MSR-SLB scaffold, after which, oneor more T-cell activating molecule(s) and T-cell co-stimulatorymolecules are loaded onto the MSR-SLB scaffold. The structures of lipidsin association with mesoporous silica microrods (MSRs) with phasecontrast microscopy, wherein a digital camera mounted on the microscopewas used to obtain images of the structures are shown in FIG. 1 . Thetop panel shows merged pictures of the lipids (green) and mesoporoussilica microrods (grey) at a lipid:MSR ratio of 1:20 (Scale=200 μm). Themiddle panel shows merged pictures of the lipids (green) and mesoporoussilica microrods (grey) at a lipid:MSR ratio of 1:4 (Scale=200 μm). Thebottom panel shows a merged phase-contrast microscope image of lipids inassociation with MSRs at a higher magnification (Scale=20 μm).

The characteristics of the antigen-presenting cell-mimetic scaffolds(APC-MS) were found to be dependent on the type of lipid and the contentof the lipid. FIG. 2A provides a list of lipids that may be used toachieve the desired architecture and/or properties of the scaffold,e.g., dioleoyl-phosphatidylcholine (DOPC);palmitoyl-oleoylphosphatidylcholine (POPC); ordistearoyl-phosphatidylcholine (DSPC). Furthermore, it was found thatthe retention of lipids layered on the MSR-SLB compositions depends onthe type and/or content of the lipid (see FIG. 2B). Next, theorganization of the lipid bilayers in the scaffolds of the invention wasstudied using fluorescence analysis. FIG. 2C shows changes in relativeflorescence of various MSR-SLB compositions containing DOPC, POPC orDSPC in phosphate-buffered saline (PBS) over a two-week (14-day) period.FIG. 2D shows changes in relative florescence of various MSR-SLBcompositions containing DOPC, POPC or DSPC in complete Roswell ParkMemorial Institute medium (cRPMI) over a two-week (14-day) period at 37°C.

Additionally, the stability of various MSR-SLB compositions at varioustime-points was investigated by suspending the scaffolds in PBS for 3days, 7 days, and 14 days. The scaffold architecture and/or structurewas then analyzed with phase-contrast fluorescence microscopy. Resultsare shown in FIG. 3 . The top panel shows the stability of DOPC in theMSR-SLB composition; the middle panel shows the stability of POPC in theMSR-SLB composition; and the bottom panel shows the stability of DSPC inthe MSR-SLB composition.

Subsequently, the assembly and the characteristics of MSR-SLB fluidstructures were studied over time with phase contrast microscopy.Results are shown in FIGS. 4A-4E. FIG. 4A shows phase-contrast confocalfluorescence microscope images of lipids in association with mesoporoussilica microrods (MSRs) taken at high magnification (scale=2 μM) priorto bleaching the lipid composition (“pre”), at the time of bleaching thelipid composition (t=0) and 5 minutes post-bleaching the lipidcomposition (t=5 min). FIG. 4B shows changes in fluorescence recoveryafter photo-bleaching (FRAP) with time. The sources are depicted inregion (2), the sinks are depicted in region (3), and the normalizationpoint is indicated as region (1). The differential distribution was bestseen at early time points after bleaching and achieved an equilibrium ataround 2 mins (120 s). The figure on the right shows smooth-fittingcurves depicting average changes in FRAP, as derived from normalizedimages, over time. FIG. 4C and FIG. 4D show two sets of high resolutionimages of MSR-SLB fluid structures prior to bleaching (pre), atbleaching (t=0) and after 3 minutes post-bleaching (t=3 min) with thelipid composition.

Furthermore, the structural and functional properties of MSR-SLBcompositions containing various lipid moieties was studied usingspectrophotometric analysis. Results are shown in FIGS. 5A and 5B. FIG.5A shows a schematic representation of MSR-SLB compositions containing alipid bilayer of POPC containing phosphoethanolamine biotin (biotin PE),which is conjugated to a streptavidin molecule (e.g., a streptavidindimer), which in turn is conjugated to a biotinylated antibody (e.g., abiotinylated anti-CD3 antibody or a biotinylated anti-CD28 antibody oranother specific or non-specific antibody). FIG. 5B showsspectrophotometric analysis of MPS (silica), POPC (lipid), MPS-POPCcomposite, biotinylated MPS-POPC composite (in the presence or absenceof streptavidin) and the MPS-POPC composite together with thebiotinylated antibody in the presence or absence of phycoerythrin biotin(biotin PE) and/or streptavidin. Significant increase in absorbance isobserved in MSR-SLB compositions containing phosphoethanolamine biotin(biotin PE) conjugated to a biotinylated antibody via a streptavidinlinker (dark bars; ** indicates statistical significance). An increasein the activity of B3Z hybridoma cells, which produce β-galactosidase inresponse to activation, was observed with all components present,indicating that APC-MS primarily adopts the structure depicted in (A).

Example 2: Analysis of the Functional Properties of the APC-MS Releaseof Homeostatic Factors Such as IL-2

APC-MS containing mesoporous silica rods (MSR) and supported lipidbilayer (SLB), which further contain IL-2 were manufactured using themethods described in Example 1. The release of IL-2 from these MSR-SLBcompositions was analyzed using staining techniques and/or bindingassays. The results are presented in FIGS. 6A and 6B. As illustrated inthe electron micrograph of FIG. 6A, high surface area pores of MSRs areavailable for potential adsorption of IL2 or other soluble payloads(scale bar=100 nm). The plot showing cumulative IL-2 release over a15-day period (FIG. 6B) shows that the APC-MS of the invention arecapable of releasing homeostatic agents such as IL-2 in a controlled andsustained manner during the entire course of the two-week study period.

Association with T-Cells

Antigen presenting cell-mimetic scaffolds containing MSR-SLB scaffolds(APC-MS) were incubated with media containing functional T-cells and theinfiltration of T-cells into the scaffolds was analyzed with phasecontrast microscopy. The results are presented in FIGS. 7A and 7B. FIG.7A shows whole cells stained with a live-cell dye and a nuclear dye. Theimage depicts live T-cells that have infiltrated into the interparticlespace of stacked high-aspect ratio lipid-coated MSR-SLB scaffolds. FIG.5B shows cells that have been stained with a single dye.

Example 3: Antibody Loading

The APC-MS containing MSR-SLB were then loaded with various stimulatorymolecules, co-stimulatory molecules and/or T-cell homeostatic agents andthe resulting structures were analyzed with fluorescence microscopy.Four different types of MSR-SLB scaffolds were analyzed—(1) nude MSR-SLBscaffold (control); (2) MSR-SLB containing conjugated antibodies; (3)MSR-SLB containing IL-2; and (4) MSR-SLB containing conjugatedantibodies and IL-2. The photomicrographs are shown in FIG. 8 (lowresolution images are on the left and high resolution images are on theright). The top panel (greyscale images) contains phase-contrastmicroscope images of each of the aforementioned MSR-SLB scaffolds. Thebottom panel merges images capturing lipid fluorescence with thegreyscale images of mesoporous silica microrods (MSR). The images on theright show MSR-SLB scaffolds at high magnification (scale bar=20 μm).

Example 4: Properties of Antibody-Loaded APC-MS

The effect of antibody-loaded APC-MS on T-cell expansion wasinvestigated using routine cytological assays. To this end, T-cells werecontacted with various control and experimental scaffolds and the effectof each on T-cell populations was measured by Alamar blue dye (indicatesmetabolic activity) and IFNγ production was measured by ELISA. Thecontrol scaffolds include shams (“mock”), SLB-free scaffolds (“free”),scaffolds containing POPC lipid only (“POPC”) and scaffolds containing acombination of POPC and IL-2. The experimental scaffolds contain acombination of POPC and IL-2, along with antibody. Three different dosesof the antibody (MSR:antibody ratio of 1:50, 1:25 and 1:10) wereinvestigated. The results are presented in FIGS. 9A and 9B. As is shownin FIG. 9A, a 3-day stimulation of T-cells with the experimentalscaffold significantly increased T-cell expansion. Moreover, the effectof the antibody on the expansion of T-cells was found to bedose-dependent. Next, an identical setup was used to analyze IFNγproduction by T-cells. Results are presented in FIG. 9B. It was foundthat incubation of T-cells in experimental scaffolds (containing POPCand IL-2 and the antibody) greatly improves IFNγ secretion compared toT-cells that were incubated in control scaffolds. Moreover, the effectof the antibody on the T-cell dependent secretion of IFNγ was found tobe dose-dependent.

The effect of the antigen-presenting cell-mimetic scaffolds (APC-MS) ofthe present invention on the expansion of metabolically-active T cellswas analyzed using routine cytometry studies. Results are presented inFIGS. 10 and 11 . In general, the scaffolds of the invention were foundto promote rapid expansion of T-cells in vitro. In this regard, FIG. 10shows fold-expansion of primary T-cells upon incubation with control orexperimental scaffolds. It was found that incubation of primary T-cellswith the compositions of the instant invention significantly inducedT-cell expansion (with or without re-stimulation) compared to mockcompositions or compositions free of SLB. More importantly, compared toa composition of DYNABEADS and IL-2, incubation of primary T-cells withthe scaffolds of the invention resulted in a measurably strongerproliferation upon re-stimulation at day 7. FIG. 11 shows a bar-chart ofmetabolic activity of T-cells (as measured by relative Alamar Blue (RFU)per cell) that were incubated with the scaffolds of the instantinvention loaded with IL-2 (SLB/IL2/ABS) or DYNABEADS loaded with IL-2(DYNABEADS-IL2). A significantly higher metabolic activity was observedin samples incubated with the scaffolds of the instant invention(left-hand columns) at day 5 and day 7 (prior to re-stimulation), andalso at day 11 (in the non-re-stimulated samples, as indicated by greenand orange bars). Re-stimulation at day 7 increased metabolic activityof both groups of T-cells i.e., those incubated with the SLB/IL2/ABScomposition or the DYNABEADS-IL2 composition compared tonon-re-stimulated cells, achieving levels that were previously observedat day 7. Re-stimulation failed to elevate mitotic activity at day 13,indicating T-cell exhaustion at this point.

The effect of the scaffolds of the instant invention on the formation ofT-cell aggregates was also studied using microscopic analysis. Resultsare presented in FIG. 12 . The images on the left-hand panel showphotomicrographs (at 4× magnification) of aggregates of splenic T cellsupon incubation with DYNABEADS or APC-MS at day 0, day 3, and day 7. Theimages on the right-hand panel show photomicrographs (at 10×magnification) of aggregates of splenic T cells upon incubation withDYNABEADS or APC-MS at day 0, day 3, and day 7. (White scale bars=100μM). It was found that the scaffolds of the invention (APC-MS) confergreater polyclonal expansion of splenic T cells (mouse) and facilitateformation of T cell aggregates than DYNABEADS.

Example 5: Use of Scaffolds to Stimulate and Expand Distinct T-CellSub-Populations

The utility of the APC-MS compositions of the invention in stimulatingand expanding specific T-cell sub-populations was performed using cellsorting techniques. Splenic T-cells were incubated with APC-MS orDYNABEADS and changes in cellular phenotype (based on expression ofcell-surface markers) were analyzed by FACS at various time-pointspost-incubation (t=0 days, 5 days, 7 days, 11 days and 13 days). In thefirst experiment, changes in the relative frequencies of CD4+ and CD8+T-cell sub-populations were analyzed using FACS, wherein the values onthe X-axis depict intensity of CD8+ staining and the values on theY-axis depict intensity of CD4+ staining. In a second experiment,polyclonal expansion of a subset of FoxP3+ mouse splenic T cells uponincubation with APC-MS or DYNABEADS was analyzed. In a third experiment,polyclonal expansion of a subset of CD62L+ mouse splenic T cells uponincubation with the scaffolds of the invention (APC-MS) or DYNABEADS. Ina fourth experiment, polyclonal expansion of a subset of CD8+/CD69+mouse splenic T cells upon incubation with APC-MS or DYNABEADS. In afifth experiment, polyclonal expansion of a subset of CD8+/Granzyme B+mouse splenic T cells upon incubation with APC-MS or DYNABEADS. In eachof the aforementioned experiments, after 7 days, a first T-cellsub-population was subject to IL-2 treatment while a second T-cellsub-population was re-stimulated and the cell suspensions were culturedfor 6 additional days. Additionally, both the APC-MS and DYNABEADS usedin the experiments were ensured to contain an identical repertoire ofstimulatory and co-stimulatory molecules.

The results of the first experiment are presented in FIGS. 13A and 13B.The results show that compared to incubation with DYNABEADS, incubationwith the scaffolds of the invention (APC-MS) achieved greater expansionof polyclonal CD8+ mouse splenic T cells at the end of the 14-dayincubation period. Also, while IL-2 treatment inhibited expansion ofcells stimulated with DYNABEADS (about 20% reduction), no such effectwas observed with cells incubated with APC-MS.

In the second experiment, a rectangular gate was applied to count thenumber and/or proportion of FoxP3+ cells in the various fractions. Theresults are presented in FIG. 14 .

In the third experiment, the results of which are shown in FIG. 15 , itwas found that the APC-MS compositions of the invention conferpolyclonal expansion of a subset of CD62L+ mouse splenic T cells in amanner that is similar and comparable to those achieved with DYNABEADS.The results are depicted in the form of flow cytometric (FACS) scatterplots of T-cell population(s) at various time-points (t=0 days, 5 days,7 days, 11 days and 13 days) following incubation with APC-MS orDYNABEADS (with re-stimulation or IL-2 treatment after 7 days ofincubation). The CD62L+ cells appear in the right hand (top and bottom)quadrants of the scatter plots. The results demonstrate that the APC-MScompositions of the invention are equally effective at selectivelyexpanding the target T cell sub-populations, which may then bemanipulated or formulated using known cytological techniques.

In the fourth experiment, the results of which are shown in FIG. 16 , itwas found that the APC-MS compositions of the invention conferpolyclonal expansion of a subset of CD8+/CD69+ mouse splenic T cells ina manner that is similar and comparable to those achieved withDYNABEADS. The results are depicted in the form of flow cytometric(FACS) scatter plots of T-cell population(s) at various time-points (t=0days, 5 days, 7 days, 11 days and 13 days) following incubation withAPC-MS or DYNABEADS (with re-stimulation or IL-2 treatment after 7 daysof incubation). The CD8+/CD69+ cells appear in the top right handquadrant of the scatter plots. The results demonstrate that compared toincubation with DYNABEADS, incubation with APC-MS achieved greaterexpansion of polyclonal CD8+/CD69+ T cells at the end of the 14-dayincubation period (relative proportion of about 90% CD8+/CD69+ T cellsin samples incubated with APC-MS versus about 50% CD8+/CD69+ T cells insamples incubated with DYNABEADS).

In the fifth experiment, the results of which are shown in FIG. 17 , itwas found that the APC-MS compositions of the invention conferpolyclonal expansion of a subset of CD8+/Granzyme B+ mouse splenic Tcells in a manner that is similar and comparable to those achieved withDYNABEADS. The results are depicted in the form of flow cytometric(FACS) scatter plots of T-cell population(s) at various time-points (t=0days, 5 days, 7 days, 11 days and 13 days) following incubation withAPC-MS or DYNABEADS (with re-stimulation or IL-2 treatment after 7 daysof incubation). The CD8+/Granzyme B+ cells appear in the top right handquadrant of the scatter plots. The results demonstrate that compared toincubation with DYNABEADS, incubation with APC-MS achieved greaterexpansion of polyclonal CD8+/Granzyme B+ T cells at the end of the14-day incubation period (relative proportion of about 95% CD8+/GranzymeB+ T cells in samples incubated with APC-MS versus about 80%CD8+/Granzyme B+ T cells in samples incubated with DYNABEADS).

Example 6: Use of Scaffolds to Stimulate and Expand Cytokine-SecretingCells

Mouse splenic T-cells were incubated for various durations (t=0 days, 5days, 7 days, 11 days and 13 days) with the APC-MS or DYNABEADS. After7-days of incubation, a first sub-population of T-cells wasre-stimulated with APC-MS or DYNABEADS, respectively, and a secondsub-population was treated with IL-2. Cytokine (IFNγ) secretion wasmeasured using standard assays for measuring IFNγ concentrations inbiological samples, e.g., ELISA assays. The results, which are presentedin FIG. 18 , demonstrate that compared to incubation with DYNABEADS,incubation with APC-MS achieved greater expansion of polyclonal CD8mouse splenic T cells after 5-days of incubation. This effect wassustained throughout the 13-day experimental period. Incubation ofsplenic T-cells with the scaffolds increased IFNγ secretion.Furthermore, it was found that re-stimulation was particularly effectivein enhancing IFNγ secretion in the sub-population of cells that wereincubated with DYNABEADS.

Example 7: Use of Scaffolds to Remove Anergeic, Quiescent or SpentT-Cells

Several new co-stimulatory molecules have been discovered based on theirhomology with the B7 and CD28 families. Programmed cell death protein 1(PD-1; UNIPROT Accession No. Q15116) is expressed on activated T cellsand has two B7 like ligands, PD-L1 and PD-L2 (Freeman et al., J. Exp.Med. 192:1027-1034 (2000); Latchman et al., Nat. Immunol. 2:261-268(2001); Dong et al., Nat. Med. 5:1365-1369 (1999); Tseng et al., J. Exp.Med. 193:839-846 (2001)). It is thought that that PD-1 is a marker ofanergy (Chikuma et al., J Immunol., 182(11):6682-9, 2009). Thus, theeffect of the scaffolds of the instant invention on inducing T-cellanergy was investigated using flow cytometry. Mouse splenic T-cells wereincubated for various durations (t=0 days, 5 days, 7 days, 11 days and13 days) with the scaffolds of the invention (APC-MS) or DYNABEADS.After 7-days of incubation, a first sub-population of T-cells wasre-stimulated and a second sub-population was treated with IL-2. EachT-cell sub-population was analyzed for expression of cell-surfacemarkers using FACS scatter plots, wherein the values on the X-axisdepict intensity of CD8+ staining and the values on the Y-axis depictintensity of PD-1+ staining. The results, which are presented in FIG. 19, show increased PD-1 expression (i.e., increased anergy) of mousesplenic T cells with time. T-cell exhaustion was achieved in bothsub-populations The results indicate that the majority of cellsthroughout culture period is PD-1 negative (lower quadrants), althoughsome cells do upregulate expression of PD-1. Restimulation with APC-MStends to increase PD-1 expression. Note: exposure to IL-2 was providedin all setups.

Example 8: Use of Scaffolds to Increase T-Cell Expansion and ImproveCell Activity

The effect of the APC-MS compositions of the invention in improvingT-cell expansion and/or metabolic activity was performed usingcytometry. Human peripheral blood T-cells were incubated with controlscaffolds or experimental scaffolds and the number and/or metabolicactivity of T-cells was measured at various time-points (t=0 days, 5days, 7 days, 11 days and 13 days) using standard assays, e.g., manualcell counts of live cells using Trypan Blue exclusion/hemocytometer,metabolic activity analyzed using Alamar Blue assay. Results arepresented in FIGS. 20A and 20B. In the case of T-cell expansion studies,control scaffolds include sham compositions (labeled: “mock”; depictedwith a black line) and compositions containing soluble, free form ofstimulants anti-CD3, anti-CD28, IL-2 (“free”; depicted with a red line),while the experimental scaffolds include (1) DYNABEADS (blue line) and(2) lipid bilayers (SLB) of the present invention (green line). Resultsare shown in FIG. 20A. It can be seen that at the end of the 13-dayexperimental period, incubation with SLB resulted in almost a two-foldgreater expansion of T-cells compared to incubation with DYNABEADS. Moresurprisingly, even the “free” scaffolds elicited a stimulatory effect onT-cells which was comparable to the effect of DYNABEADS.

Results on the effect of the scaffolds of the instant invention on themetabolic activity of primary T-cells are presented in FIG. 20B. SplenicT-cells were incubated with control scaffolds or experimental scaffoldsand the metabolic activity of T-cells was measured at varioustime-points (t=0 days, 5 days, 7 days, 11 days and 13 days) was measuredusing Alamar Blue staining. The control scaffolds include shamcompositions (“mock”; “m”) and compositions that are free of SLB(“free”; “f”), while the experimental scaffolds include (1) DYNABEADS(“d”) and (2) lipid bilayers (SLB) (“s”). It can be seen that at the endof the 14-day experimental period, cells incubated with “mock” scaffoldsall perish, while cells incubated with the experimental scaffolds (SLBor DYNABEADS) experience sustained growth and expansion over time. Moreimportantly, the SLB scaffolds of the invention promoted better growthand metabolic activity of T-cells at the end of the 14-day experimentalperiod compared to the effects conferred by DYNABEADS.

Example 9: Use of Scaffolds to Promote Expansion of Human T-Cells

Human blood samples obtained from subject 1 (FIG. 21A) and subject 2(FIG. 21B) were incubated with control scaffolds (“mock”) orexperimental scaffolds containing the listed anti-CD3antibodies—muromonab (OKT3), an antibody recognizing 17-19 kDa ε-chainof CD3 within the CD3 antigen/T cell antigen receptor (TCR) complex(HIT3a) and a monoclonal antibody recognizing a 20 kDa subunit of theTCR complex within CD3e (UCHT1). Three different dosages wereinvestigated—5 μg (top slides), 1 μg (bottom slide for subject 2) and0.5 μg (bottom slide for subject 1). In each case, co-stimulation wasprovided with anti-CD28 antibodies, wherein the ratio of anti-CD3antibody:anti-CD28 antibody was maintained at 1:1. Fold expansion of Tcells was measured at various time-points (t=0 days, 7 days, 11 days and13 days). The results, which are presented in FIGS. 21A and 21B, showthat at higher antibody dosages (5 μg), all three anti-CD3 antibodieswere capable of stimulating expansion of human T-cells. In all cases,the expansion of T-cell population was initially slow until day 7, afterwhich, it increased exponentially. At the highest dose, a 600-800 foldincrease in the number of T-cells was achieved at the end of theexperimental period (day 13). With intermediate dosage (1 μg), only OKT3and HIT3a (but not UCHT1) were capable of stimulating T-cell expansion,wherein, a 300-400-fold increase in the number of T-cells was achievedat the end of the experimental period (day 13). At the lowest dosage(0.5 μg), only OKT3 (but not UCHT1 and/or HIT3a) was capable ofstimulating T-cell expansion, wherein, a 600-700 fold increase in thenumber of T-cells was achieved at the end of the experimental period(day 13). The results show an effect of both the anti-CD3 antibody cloneas well as dose of the antibody on the expansion rate.

Next, the polyclonal expansion of a human T cells upon incubation withcontrol scaffolds (“mock”) or experimental scaffolds containing thelisted anti-CD3 antibodies—OKT3, HIT3a, and UCHT1, was analyzed via flowcytometry. Results are presented in FIG. 22 , wherein the bottom panelsshow flow cytometric (FACS) scatter plots of T-cell population(s) atvarious time-points (t=8 days, 11 days and 14 days) following incubationwith APC-MS containing each of the anti-CD3 antibodies as a stimulatorymolecule and an anti-CD28 antibody as the T-cell co-stimulatorymolecule. The values on the X-axis of the scatter plots depict intensityof CD8+ staining and the values on the Y-axis depict intensity of CD4+staining. The scatter plots are summarized in the line-graphs of the toppanel, which show changes in percentage of CD4+ versus CD8+ T-cellsub-populations after incubation with APC-MS containing theaforementioned anti-CD3 antibodies—OKT3 (circles), HIT3a (squares) andUCHT1 (triangles). Two different antibody dosages were studied-a firstdose of 5 μg (1× dilution) and a second dose of 0.5 μg (1:10× dilution).The results show that at low antibody dosages (1:10× dilution; 0.5 μg),all three anti-CD3 antibodies were capable of enriching CD8+-specificT-cells using a low antibody concentration versus a high antibodyconcentration. A 3-4 fold increase in the number of CD8+-specificT-cells was achieved at the end of the experimental period (day 14). Forinstance, the relative frequency of CD8+ T-cells was 20% at the start ofthe experiment, which had increased to about 60%-80% at day 14.Moreover, it was found that anti-CD3 antibodies UHCT1 and OKT1 wereequally effective and superior to the anti-CD3 antibody HIT3a inpromoting the expansion of CD8+ T-cells. At high (5 μg) antibody doses,the ratio of CD8+:CD4+ in the global T-cell population was unchanged (oreven attenuated) at day 14. The results show an effect of both theanti-CD3 antibody clone as well as dose of the antibody on the expansionof CD8+-specific T cells.

Example 10: Use of Scaffolds to Promote Expansion of a Specific HumanT-Cell Sub-Population

Human blood samples were incubated with experimental scaffoldscontaining the listed anti-CD3 antibodies—muromonab (OKT3), HIT3a andUCHT1 at 1X dosage (5 μg). In each case, co-stimulation was providedwith anti-CD28 antibodies. Fold expansion of T cells was measured after14 days. The expression of CD62L and CCR7 in total live cells is shownin the top panels and the expression of these markers in gated CD8+cells is shown in the bottom panels. The results, which are presented inFIG. 23 , show that all three anti-CD3 antibodies were capable ofstimulating the expansion of a distinct sub-population of human T-cells.Surprisingly, a majority of cells expanded with the variousantigen-presenting cell-mimetic scaffolds (APC-MS) of the instantinvention remain CD62L+CCR7+ even after 14 days post-incubation. Theresults point to the retained in vivo functionality of expanded T-cellsafter ex vivo expansion. Accordingly, it is possible to selectivelyexpand and sustain a distinct sub-population of CD62L+CCR7+ T-cells(e.g., memory cells) using the antigen-presenting cell-mimeticscaffolds.

Additionally, APC-MS scaffolds containing OKT3 were particularlyeffective in expanding and/or retaining CD62L+CCR7+ T-cells compared toscaffolds containing UCHT1 and/or HIT3a.

Example 11: Expansion of T-Cells Ex Vivo Using Antigen-PresentingCell-Mimetic Scaffolds (APC-MS)

Adoptive cell transfer (ACT) of T cells is a promising treatment forcancer and infectious disease. However, current approaches for ex vivo Tcell expansion, a key step in ACT, frequently yield suboptimal expansionrates and limited functionality of cells. Here, we developed mesoporoussilica micro-rod-supported lipid bilayers that presented cues for T cellreceptor stimulation and costimulation at predefined densities locallyon a fluid lipid bilayer, and facilitated the controlled release ofsoluble interleukin-2, similar to how these cues are naturally presentedby antigen-presenting cells (APCs). In cell culture, the material formedinto an APC-mimetic scaffold (APC-MS) that promoted the activation ofinfiltrating mouse and human T cells. APC-MS promoted two- to ten-foldgreater polyclonal T cell expansion than commercial expansion beadsafter two weeks, and robust antigen-specific expansion of raresubpopulations of functional cytotoxic T cells. This study demonstratesa new platform to rapidly expand functional T cells for ACT.

Adoptive cell transfer (ACT) using T cells is a promising approach forthe treatment of various malignancies and infectious diseases (see e.g.,Rosenberg, S. A. & Restifo, N. P. Science 348, 62-68 (2015); June, C. H.et al. Science Translational Medicine 7(280): 280ps7 (2015); and Fesnak,A. D. et al. Nature Reviews Cancer 16, 566-581 (2016)). However, therapid ex vivo expansion of functional T cells, a key step in theproduction of T cells for ACT, remains a major challenge. T cellactivation requires three signals: (1) T cell receptor (TCR)stimulation, (2) costimulation, and (3) pro-survival cytokines (Huppa,J. B. & Davis, M. M. Nature Reviews Immunology 3, 973-983 (2003)). Inthe body, these signals are provided by antigen-presenting cells (APCs),which present these cues to T cells in specific spatiotemporal patterns(Huppa and Davis (2003); Lee, K.-H. et al. Science 302, 1218-1222(2003); Alarc6n, B. et al. Immunology 133, 420-425 (2011); and Minguet,S. et al. Immunity 26, 43-54 (2007)). Various approaches are used toexpand T cells ex vivo for ACT, and synthetic artificial APCs (aAPCs)are particularly convenient (Rosenberg and Restifo (2015); Hasan, A. etal. Advancements in Genetic Engineering 2015 (2015); Hollyman, D. et al.Journal of Immunotherapy (Hagerstown, Md.: 1997) 32, 169 (2009); Maus,M. V. et al. Nature Biotechnology 20, 143-148 (2002); Zappasodi, R. etal. Haematologica 93, 1523-1534 (2008); Perica, K. et al. ACS Nano 9,6861-6871 (2015); Mandal, S. et al. ACS Chemical Biology 10, 485-492(2014); Steenblock, E. R. & Fahmy, T. M. Molecular Therapy 16, 765-772(2008); Fadel, T. R. et al. Nature Nanotechnology 9, 639-647 (2014);Sunshine, J. C. et al. Biomaterials 35, 269-277 (2014); Fadel, T. R. etal. Nano letters 8, 2070-2076 (2008); Meyer, R. A. et al. Small 11,1519-1525 (2015); and Steenblock, E. R. et al. Journal of BiologicalChemistry 286, 34883-34892 (2011)). Currently, commercial microbeads(DYNABEADS) functionalized with activating antibodies for CD3 (αCD3; TCRstimulus) and CD28 (aCD28; costimulatory cue) are the only FDA-approvedsynthetic system for expanding T cells (Hollyman et al. (2009)). Thesebeads promote polyclonal T cell activation with exogenous interleukin-2(IL-2) supplementation. Although these cultures provide T cells with thethree critical signals, the context in which these signals are presentedis not representative of how they are naturally presented by APCs. Thiscontextual inconsistency can lead to suboptimal T cell expansion ratesand T cell products with limited or dysregulated functions (Zappasodi etal. (2008); Fadel et al. (2014); L1, Y. & Kurlander, R. J. Journal ofTranslational Medicine 8, 1 (2010); and JJin, C. et al. MolecularTherapy-Methods & Clinical Development 1 (2014)). In addition, thesebeads are not amenable to the presentation of large sets of cues, whichmay be important for the generation of highly functional therapeutic Tcells (Hasan et al. (2015); and Hendriks, J. et al. Nature immunology 1,433-440 (2000)).

A composite material was developed comprised of supported lipid bilayers(SLBs) formed on high aspect ratio mesoporous silica micro-rods (MSRs)(Kim, J. et al. Nature Biotechnology 33, 64-72 (2015); and Li, W. A. etal. Biomaterials 83, 249-256 (2016)). The SLBs enabled the presentationof combinations of T cell activation cues at predefined densities on afluid lipid bilayer, while the MSRs facilitated the sustained paracrinerelease of soluble cues to nearby T cells. Thus, composite MSR-SLBsenabled the presentation of surface and soluble cues to T cells in acontext analogous to natural APCs. In cell culture, the high aspectratio rods settled and stacked to form a 3D scaffold structure. Thescaffolds formed from MSR-SLBs that were functionalized with T cellactivation cues are referred to as APC-mimetic scaffolds (APC-MS).APC-MS facilitated between two- to ten-fold greater polyclonal expansionof primary mouse and human T cells than commercial DYNABEADS after twoweeks. APC-MS also facilitated robust antigen-specific expansion offunctional mouse and human cytotoxic T cells. In particular, APC-MSpresenting Epstein bar virus (EBV)-associated antigens expanded andenriched for rare subpopulations of human T cells in an antigen-specificmanner. Overall, APC-MS represents a flexible and tunable platformtechnology that could enable the rapid expansion of highly functional Tcells for ACT. specific spatiotemporal patterns (Huppa and Davis (2003);Lee et al. (2003); Alarc6n et al. (2011); and Minguet et al. (2007)).

Assembly and Characterization of APC-MS

APC-MS were prepared for T cell activation (FIG. 25A), using unique cuesfor polyclonal and antigen-specific expansion (FIG. 25B). High aspectratio MSRs with average dimensions of 88 μm length, 4.5 μm diameter(aspect ratio ˜20), and 10.9 nm pores were synthesized as previouslydescribed (Kim et al. (2015); and Li et al. (2016)) (see FIG. 26A), andadsorbed with IL-2.

Liposomes (140 nm) containing predefined amounts of a biotinylated-lipidwere prepared, and coated onto the 1L-2-laden MSRs, forming MSR-SLBs(see FIG. 26B). Next, biotinylated cues for TCR activation andcostimulation were attached to the MSR-SLB surfaces via a streptavidinintermediate. In cell culture, 3D scaffolds spontaneously formed throughthe settling and random stacking of the rods, forming APC-MS. T cellsinfiltrated the interparticle space of the scaffolds. Together,scaffolds present cues for TCR-activation and costimulation on thesurface of the lipid bilayer, and release soluble IL-2 over time in aparacrine fashion to infiltrating T cells, similar to how these cues arepresented to T cells by natural APCs (see Huppa and Davis (2003)).

MSRs were coated with the phospholipid1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), which iscommonly used as a model for mammalian cell membranes (Jerabek, H. R. etal. Journal of the American Chemical Society 132, 7990-7 (2010); andTorres et al. Lab on a Chip 13, 90-99 (2013)). At low lipid:MSR ratios,lipid-mediated aggregation of MSRs was observed (FIG. 27A), while athigher lipid:MSR ratios, lipid-coated MSRs were maintained in a welldispersed, single-particle state (FIG. 27B). At this higher lipid:MSRratio, 34.1±0.9% of the input POPC was initially associated with theMSRs, and the POPC coating was slowly lost over time in cell cultureconditions (FIG. 28A) as the POPC-coated MSRs degraded (FIG. 28B). MSRswere also successfully coated with1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) and1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC). The amount of lipidassociated with MSRs was inversely related to the saturation of thelipid, likely due to tighter packing of more highly saturated lipids inthe lipid bilayers. No significant differences were observed in thestability of the various lipid coatings. To evaluate whether MSR lipidcoatings were continuous, fluid SLB structures, fluorescence recoveryafter photobleaching (FRAP) studies were carried out using afluorophore-tagged lipid. Recovery of fluorescence at photobleachedregions of lipid-coated MSRs and coincident normalization offluorescence across bleached rods was observed, demonstrating that theMSR lipid coatings were continuous, fluid SLBs (FIGS. 39A and 39B).

The loading and release of soluble cues, and the loading of surfacecues, were also analyzed. MSRs have a very high surface area availablefor surface adsorption of molecular payloads (Kim et al. (2015)), andwhen 500 μg of MSRs were loaded with 2 μg of IL-2 (0.04 mg/ml IL-2),50±1% of the input IL-2 was retained with the MSRs. The loaded IL-2 wassubsequently released in a controlled manner over 9 days. The trendcould be well approximated using a one phase exponential function(R2=0.98), indicating that the release of IL-2 followed first-orderkinetics (FIG. 28E).

The attachment of surface cues as the amount of the biotinylated lipidspecies incorporated into the lipid formulation was varies was alsoanalyzed. Streptavidin was added at 30% of the molar amount ofbiotinylated lipid groups on the respective MSR-SLB formulations, andbiotinylated IgG was added as a surface cue proxy. At saturation, themaximal amount of biotinylated IgG that could be loaded onto the variousMSR-SLB formulations differed by a factor of ˜10 (FIG. 28F). Thisdifference is consistent with the relative differences in the amounts ofbiotinylated lipid in the various MSR-SLB formulations, indicating thatthe density of surface-bound IgG could be precisely controlled bydefining the amount of adhesive lipid in the coating lipid formulation.In all subsequent experiments, MSR-SLBs were saturated with surface cuesas described, and relative surface cue density is described by the mol %of biotinylated lipid in the formulation.

To confirm that the presentation of activation cues on the scaffoldsurface promoted T cell interactions, primary T cells were culturedeither with MSR-SLBs without surface T cell cues, or complete APC-MS.Whereas T cells largely ignored MSR-SLBs without surface T cell cues,they interacted robustly with APC-MS, reorganizing the structure of thescaffolds to form extensive, high density cell-material clusters (FIG.28G and FIG. 29 ).

Polyclonal Expansion of Primary Mouse and Human T Cells

Primary mouse T cells were cultured with either DYNABEADS or withAPC-MS. Culture with APC-MS led to the formation of large cell-materialclusters, and the size and frequency of these clusters was greater inAPC-MS cultures than in Dynabead cultures (FIG. 30A). Culture withAPC-MS promoted more than two-fold greater expansion than culture withDYNABEADS (FIG. 30B).

Interestingly, whereas DYNABEADS promoted moderate CD8-biased skewing ofthe T cell population over the culture period, APC-MS promoted greaterthan 95% of total T cells being CD8+(FIG. 30C and FIG. 31 ). EffectorCD8+ T cells expanded using APC-MS upregulated the cytotoxic mediatorGranzyme B more rapidly and to a greater extent over the culture periodthan did CD8+ T cells expanded with DYNABEADS (FIG. 32A). In bothDynabead- and APC-MS-expanded T cell products, no expansion of CD4+FoxP3+ cells was observed (FIG. 32B). Importantly, despite the rapidexpansion rate observed, the majority of APC-MS-expanded T cellsremained negative for the exhaustion marker PD-1 (FIG. 32C).

APC-MS formulations were also evaluated for the polyclonal expansion ofprimary human T cells. Culture of primary human T cells with APC-MS alsoled to the formation of large cell-material clusters, with the size andfrequency of these clusters being greater in APC-MS cultures than inDynabead cultures. The stability and persistence of these clusters wasobserved to be dependent on both surface cue density and initialmaterial input (FIG. 30D). Culture for 14 days with all of the testedAPC-MS formulations led to between two- to ten-fold greater expansionthan with DYNABEADS (FIG. 30E). Interestingly, APC-MS formulationscontaining higher amounts of T cell stimuli, either via a higher surfacecue density or higher mass of initial material, promoted extremeCD4-biased skewing after 14 days of culture. In contrast, the APC-MSformulation that contained a lower overall amount of T cell stimulirelative to the other APC-MS formulations (F4), promoted a more balancedCD4+ and CD8+ expansion, comparable to the DYNABEADS (FIG. 30F). Amongthe APC-MS formulations tested, a positive correlation was observedbetween the total amount of T cell stimuli in the formulation and thefrequency of cells that co-expressed the exhaustion markers PD-1 andLAG-3 at the end of the culture period. Strikingly, despite nearly a10-fold greater expansion over a two-week culture period, lowfrequencies of PD-1 and LAG-3 co-expressing cells (<5%) was observedwith the low T cell stimuli APC-MS formulation (F4), similar toDYNABEADS. However, a higher frequency of cells co-expressing PD-1 andLAG-3 was observed in the DYNABEADS condition at day 7 (FIG. 30G). Nosignificant differences were observed between Dynabead- orAPC-MS-expanded T cell products in the frequency of cells thatco-expressed the lymphoid homing molecules CCR7 and CD62L (FIG. 33 ),which indicate a more naive T cell phenotype and have been shown to beimportant for function after in vivo transfer (Gattinoni, L. et al. TheJournal of Clinical Investigation 115, 1616-1626 (2005)). Together,these data show that APC-MS were capable of polyclonally expanding mouseand human T cells more rapidly than DYNABEADS.

Antigen-Specific Expansion of Primary Mouse T Cells

To determine whether APC-MS could be adapted for antigen-specificexpansion using primary mouse CD8+ T cells isolated from OT-I mice,which express a TCR specific for the SIINFEKL (SEQ ID NO: 4) peptidefrom chicken ovalbumin in the context of H-2K(b) MHC class I. Minimalcell-material interactions were observed when these cells were culturedwith an APC-MS formulation presenting an irrelevant peptide-loaded MHC(pMHC). However, when the cells were cultured with an APC-MS formulationpresenting SIINFEKL (SEQ ID NO: 4), robust interactions resulting in theformation of extensive cell-material clusters was observed (FIG. 34A).APC-MS formulations presenting SIINFEKL (SEQ ID NO: 4) promoted robustexpansion of OT-I CD8+ T cells, even with surface cues presented on aslow as 0.01 mol % of the lipids (FIG. 34B). In response to SIINFEKL (SEQID NO: 4) presentation from B16-F10 melanoma cells, the expanded T cellssecreted IFN-γ (FIG. 34E), upregulated the co-expression of IFN-γ andTNFα (FIG. 34C), and killed target cells in vitro (FIG. 34D).

Antigen-Specific Expansion of Primary Human T Cells

To determine whether APC-MS could be used for the antigen-specificenrichment and expansion of rare human T cell subpopulations, whichcould be useful for the selective expansion of rare cancerantigen-specific T cells from tumors or blood (Cohen, C. J. et al. TheJournal of Clinical Investigation 125, 3981-3991 (2015); and Streinen,E. et al. Science 352, 1337-1341 (2016)). APC-MS formulations presentedone of two peptides (abbreviated either CLG or GLC), from differentEBV-associated proteins, in the context of the HLA-A2 allotype of MHCclass I. CD8+ T cells were isolated from human blood samples fromHLA-A2-matched donors with prior EBV exposure, and treated with eithersoluble IL-2 (30 U/ml) alone (mock), or cultured with APC-MS presentingeither the CLG or GLC peptide. Robust antigen-specific enrichment andexpansion of the two T cell subsets was observed, while a minimalincrease in total T cells was noted (FIG. 35A). The frequency ofCLG-specific CD8+ T cells increased from 0.04% of all CD8+ T cells atday 0, to 3.3±0.9% of CD8+ T cells at day 14 when cultured withCLG-presenting APC-MS (FIGS. 36A and 36B), corresponding to a170±70-fold expansion in cell number (FIG. 36C). Similarly, thefrequency of GLC-specific CD8+ T cells increased from 0.66% of all CD8+T cells at day 0, to 48±9% at day 14 when cultured with GLC-presentingAPC-MS (FIGS. 36D and 36E), corresponding to a 300±100-fold expansion incell number (FIG. 36F). The functionalities of the various T cellproducts were analyzed in co-culture experiments with T2 stimulatorcells by evaluating IFN-γ secretion (FIG. 35B), IFNγ and TNFαco-expression (FIG. 35C, and FIGS. 36G, 36H, and 36I), and the in vitrokilling of peptide-loaded target cells (FIG. 36J). CD8+ T cellpopulations expanded with either CLG or GLC-presenting APC-MS respondedstrongly to stimulator cells that presented their cognate antigen.Notably, following co-culture with T2 cells, the frequency of CLG- andGLC-specific cells detected via tetramer staining was similar to thefrequency of cells that co-expressed IFN-γ and TNFα, indicating that themajority of the expanded T cells were functional.

To determine whether antigen-specific T cells could be expanded directlyfrom heterogeneous cell populations, such as PBMCs, obviating the needfor T cell isolation the following experiments were performed. PBMCsamples from BLA-A2-matched donors with prior EBV exposure were culturedwith a GLC-presenting APC-MS formulation. Remarkably, the frequency ofGLC-specific T cells increased from 0.66% of total CD8+ T cells at day0, to 15±1% at day 7; minimal changes were found in mock-treated samples(FIG. 36K). This corresponds to a 60±9-fold expansion of GLC-specific Tcells (FIG. 36L). The functionality of the expanded T cells wasevaluated by co-culturing with T2 cells that were either unpulsed, orpulsed with the CLG or GLC peptide. Quantification of the frequency ofcells co-expressing TNFα and IFN-γ (FIG. 36M), and 1FN1 secretion (FIG.36N), demonstrated that CD8+ T cell populations that were expanded fromPBMCs with GLC-presenting APC-MS responded robustly only to T2 cellsthat presented their cognate antigen. Taken together, these datademonstrate the ability of APC-MS to robustly expand both mouse andhuman T cells in an antigen-specific manner.

To determine whether the improvements observed using APC-MS overDYNABEADS were not solely attributable to differences in the amount ofanti-CD3 antibody and anti-CD28 antibody presented, the amount ofanti-CD3 and anti-CD28 antibodies in the DYNABEADS was normalized tocorrespond to the concentration of these antibodies in the APC-MSs. Asshown in FIGS. 40B, 40C and 40D, when the amount of anti-CD3 andanti-CD28 antibodies present in the APC-MS and DYNABEADs was matched,APC-MS promoted more rapid expansion of primary mouse T cells (FIG. 40B)while maintaining comparable co-expression levels of the exhaustionmarkers PD-1 and LAG-3 (FIG. 40C). Also, by tuning the APC-MSformulation, the CD4:CD8 ratio can be tuned (FIG. 40D).

IL-2 was observed to be released from APC-MS in a sustained manner overthe course of approximately one week. To evaluate the effect of IL-2dose and sustained release from APC-MS, primary mouse T cells werecultured for 7 days with either DYNABEADs or APC-MS presenting the sameamount of anti-CD3 and anti-CD28 antibodies. For APC-MS conditions, IL-2was either loaded onto the APC-MS and allowed to release over time(M-D), or the same dose of IL-2 was added as a soluble bolus into themedia on d0 (M-D/bIL-2). For DYNABEAD conditions, IL-2 was eithersupplemented in the media at the manufacturer recommended dose andrefreshed at each media change (D-B), or added as a soluble bolus intothe media on d0 at the same dose as was loaded into APC-MS (D-B/bIL-2).As shown in FIG. 41A, APC-MS promoted greater expansion of primary mouseT cells when IL-2 was loaded into the APC-MS and allowed to be releasedover time than when the same dose of IL-2 was added into the media as asoluble bolus, demonstrating the benefit of presenting IL-2 in thiscontext. APC-MS promoted greater expansion of primary mouse T cells thanDYNABEADs when the amounts of anti-CD3, anti-CD28 and IL-2 presented arematched (M-D/bIL-2 vs D-B/eIL-2) demonstrating the benefit of presentingthese cues in the context of APC-MS. As shown in FIG. 41B, when theamounts of anti-CD3, anti-CD28 and IL-2 presented are matched, T-cellsexpanded with APC-MS showed lower co-expression of the exhaustionmarkers PD-1 and LAG-3 than those expanded with DYNABEADs (M-D/bIL-2 vsD-B/bIL-2).

The experiments above demonstrate that the APC-MS are a multifunctionalmaterial can present TCR stimuli and costimulatory cues locally on thesurface of a fluid lipid bilayer, and facilitate the sustained,paracrine delivery of soluble cytokines to nearby T cells. Ternaryformulations presenting αCD3 or pMHC, αCD28, and IL-2 promoted rapidpolyclonal and antigen-specific expansion of primary mouse and human Tcells, including significantly faster polyclonal expansion thancommercial DYNABEADS. Importantly, despite the increased expansion rateobserved with the APC-MS used in this example, expanded T cells couldretain a functional phenotype, demonstrating that expansion rate is notfundamentally inversely coupled to function. T cells largely ignored theAPC-MS unless they were formulated to present relevant TCR cues, whichallowed for specific expansion of rare subpopulations of T cells evenfrom complex cell mixtures, such as PBMCs.

The results of these studies support the importance of presenting bothsurface and soluble cues to T cells in a manner that is comparable tohow these cues are naturally presented. Prior work on synthetic aAPCshave demonstrated that delivering cytokines such as IL-2 to T cells in aparacrine manner can potentiate the effects of the cytokine (Steenblockand Fahmy (2008); and Fadel et al. (2014)). Current systems primarilyfocus on enhancing T cell activation through the static high densitypresentation of stimuli to promote TCR clustering (Zappasodi et al.(2008); Fadel et al. (2014); and Fadel et al. (2008)). However, theclustering of TCRs is only one step in a dynamic process involving thereorganization of many cell surface molecules over time that serves notonly to enhance T cell activation, but also to limit the duration of TCRsignaling in order to protect against T cell overstimulation (Huppa andDavis (2003); Lee et al. (2003); Alarcón et al. (2011)). When presentingT cell cues across the surface of a fluid lipid bilayer, emulating howthese cues are naturally encountered on the surface of APC plasmamembranes, relatively lower surface cue densities were observed topromote more rapid expansion rates and generated T cells with a morefunctional and less exhausted phenotype.

Very high aspect ratio particles were used to form APC-MS, which is incontrast to most previously described synthetic aAPC materials(Steenblock and Fahmy (2008); Fadel et al. (2014); Sunshine et al.(2014); Fadel et al. (2008); Meyer et al. (2015); and Steenblock(2011)). These particles spontaneously settled and stacked to form highsurface area, 3D structures, which infiltrating T cells remodeled toform dense cell-material clusters, creating a microenvironment in whichT cells are in close proximity to the material. This likely allows formore efficient paracrine delivery of IL-2, and increased T cell-T cellparacrine signaling (Long, M. & Adler, A. J. The Journal of Immunology177, 4257-4261 (2006)). The relatively large size and high aspect ratioof the rods likely contributed to the formation of the larger clustersobserved in APC-MS versus Dynabead cultures, since many more T cellscould interact with each rod than with the smaller spherical DYNABEADS.The persistence of these clusters in APC-MS cultures was dependent onsurface cue density and the amount of material in the culture, whichlikely contributed to the different phenotypes observed in the variousAPC-MS conditions.

In polyclonal mouse T cell expansion studies, APC-MS promoted extremeCD8-biased skewing of the T cell population. This is consistent withprevious observations that paracrine delivery of IL-2 enhanced theproliferation of mouse CD8+ T cells, but promoted activation-inducedcell death in mouse CD4+ T cells (Steenblock et al. (2011)). However, inpolyclonal human T cell expansion studies, skewing was dependent on theoverall amount of T cell stimuli presented by the APC-MS, withconditions containing higher amounts of T cell stimuli promoting extremeCD4-biased skewing. This discrepancy could indicate fundamentaldifferences in how mouse and human T cells respond to these cues. Abetter understanding of this behavior could enable material formulationsthat bias mixed T cell populations toward specific CD4:CD8 ratios, aproperty that has recently been shown to be important for the functionof adoptively transferred T cells (Turtle, C. J. et al. The Journal ofClinical Investigation 126 (2016)).

The need to rapidly generate therapeutically relevant numbers offunctional T cells ex vivo is a significant challenge in personalized Tcell therapies, and the results of this study indicate that APC-MSprovides a significant advancement towards meeting this need (Turtle, C.J. & Riddell, S. R. Cancer Journal (Sudbury, Mass.) 16, 374 (2010); andEggermont, L. J. et al. Trends in Biotechnology 32, 456-465 (2014)). Asingle stimulation with ternary APC-MS formulations was observed topromote significantly faster T cell expansion than commercial DYNABEADS,and demonstrated that parameters of the material could be manipulated toimprove the phenotype of the cell product without compromising the rapidexpansion rate. As APC-MS is a modular platform technology, componentsof the system can be altered or changed to modify the spatial andtemporal context in which cues are presented. For example, altering MSRproperties may allow for tuning of the scaffold microenvironment ordegradation kinetics. Changing the lipid formulation may enable tuningof SLB stability, fluidity, or surface cue partitioning, or theattachment of cues via different chemistries (Torres et al. (2013); Puu,G. & Gustafson, I. Biochimica et Biophysica Acta (BBA)-Biomembranes1327, 149-161 (1997); Anderson, N. A. et al. Journal of the AmericanChemical Society 129, 2094-2100 (2007); Collins, M. D. & Keller, S. L.Proceedings of the National Academy of Sciences 105, 124-128 (2008);Reich, C. et al. Biophysical Journal 95, 657-668 (2008); Longo, G. S. etal. Biophysical Journal 96, 3977-3986 (2009); Kwong, B. et al.Biomaterials 32, 5134-5147 (2011); Koo, H. et al. Angewandte ChemieInternational Edition 51, 11836-11840 (2012); and Desai, R. M. et al.Biomaterials 50, 30-37 (2015)). The APC-MS described herein may also bealtered to present larger sets of both surface and soluble cues, whichmay enable the generation of further optimized T cells for ACTS (Hasanet al. (2015); and Hendriks et al. (2000)).

Methods Cells and Reagents

The B16-F10 murine melanoma cell line was obtained from ATCC, andconfirmed to be negative for mycoplasma. B16-F10 cells were cultured inDulbecco's modified Eagle's medium (DMEM) supplemented with 10%heat-inactivated fetal bovine serum (FBS) (HI-FBS) and 1%penicillin-streptomycin. The B3Z murine T cell hybridoma cells werecultured in RPMI 1640 supplemented with 10% HI-FBS, 2 mM L-glutamine, 1mM sodium pyruvate, 50 μM beta-mercaptoethanol, and 1%penicillin-streptomycin. The T2 (174×CEM.T2) human lymphoblast cellswere cultured in RPMI 1640 supplemented with 10% HI-FBS, 2 mML-glutamine, 1 mM sodium pyruvate, 50 μM beta-mercaptoethanol, 0.1 mMnon-essential amino acids, 1 mM sodium pyruvate, 10 mM HEPES, and 1%penicillin-streptomycin. Primary mouse and human T cells were culturedin RPMI 1640 supplemented with 10% HI-FBS, 2 mM L-glutamine, 1 mM sodiumpyruvate, 50 μM beta-mercaptoethanol, 0.1 mM non-essential amino acids,1 mM sodium pyruvate, 10 mM HEPES, and 1% penicillin-streptomycin,supplemented with 30 U/ml recombinant mouse- or human-IL-2,respectively.

All chemical reagents for MSR synthesis were purchased fromSigma-Aldrich. All lipids were purchased from Avanti Polar Lipids.Specific lipids used in these studies are as follows: DOPC (850375C),POPC (850457C), DPSC (850365C), PE-cap-biotin (870273C), 18:1PE-carboxyfluorescein (810332C). FoxP3 antibodies were purchased fromeBioscience. All other antibodies were purchased from Biolegend. Murineand human recombinant IL-2 were purchased from Biolegend. Biotinylatedpeptide-loaded MHC monomers and fluorophore-labeled tetramers wereobtained from the National Institutes of Health Tetramer Core Facility.Mouse and human CD3/CD28 T cell expansion DYNABEADS were purchased fromThermoFisher Scientific. The ovalbumin-derived peptide SIINFEKL (SEQ IDNO: 4) was purchased from Anaspec. The EBV-derived peptides CLGGLLTMV(SEQ ID NO: 1) and GLCTLVAML (SEQ ID NO: 2) were purchased fromProimmune.

Synthesis of Mesoporous Silica Micro-Rods (MSRs)

MSRs were synthesized as previously reported (Kim et al. (2015); and Liet al. (2016)). Briefly, 4 g of Pluronic P123 surfactant (averageMn-5,800, Sigma-Aldrich) was dissolved in 150 g of 1.6 M HCl solutionand stirred with 8.6 g of tetraethyl orthosilicate (TEOS, 98%,Sigma-Aldrich) at 40° C. for 20 h, followed by aging at 100° C. for 24h. Subsequently, surfactant was removed from the as-prepared particlesby extraction in 1% HCl/ethanol (v/v) at 70° C. for 20 hours. Particleswere recovered by running the suspension through a 0.22 μm filter,washed with ethanol, and dried.

Primary Mouse T Cell Isolation

All procedures involving animals were done in compliance with NationalInstitutes of Health and Institutional guidelines. Animals werepurchased from The Jackson Laboratory. For polyclonal T cell expansionstudies, C57BL/6J mice were used as cell donors. For antigen-specific Tcell expansion studies, C57BL/6-Tg(TcraTcrb)1100Mjb/J (OT-I) mice wereused as cell donors. All animals were female and used between 6 and 9weeks old at the start of the experiment. To isolate T cells,splenocytes were prepared by mashing spleens through 70 μm nylon cellstrainers, and red blood cells were lysed in ACK buffer. Subsequently,either CD3+ T cells were isolated for polyclonal T cell expansionstudies using a pan T cell isolation MACS kit (Miltenyi Biotec), or CD8+T cells were isolated for antigen-specific T cell expansion studiesusing a CD8a+ T cell isolation MACS kit (Miltenyi Biotec).

Primary Human T Cell Isolation

De-identified leukoreduction collars were obtained from the Brigham andWomen's Hospital Specimen Bank. PBMCs were isolated from leukoreductionsin a Ficoll gradient, followed by two washes to remove plateletcontaminants. Subsequently, in some studies, either CD3+ T cells wereisolated for polyclonal T cell expansion studies using a pan T cellisolation MACS kit (Miltenyi Biotec), or CD8+ T cells were isolated forantigen-specific T cell expansion studies using a CD8+ T cell isolationMACS kit (Miltenyi Biotec).

Preparation of Antigen-Presenting Cell-Mimetic Scaffolds (APC-MS)

MSRs and liposomes were prepared prior to APC-MS assembly. To prepareliposomes, lipid films composed of predefined lipid formulations werefirst prepared by mixing lipid-chloroform suspensions, evaporating thebulk chloroform under nitrogen, and removing residual chloroformovernight in a vacuum chamber. For all functional studies,1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) was used as theprimary lipid, and lipid formulations were doped with between 0.01-1 mol% of either the carboxyfluorescein-tagged lipid1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-(carboxyfluorescein), orthe biotinylated lipid1,2-di-(9Z-octadecenoyl)-sn-glycero-3-phosphoethanolamine-N-(capbiotinyl). For some characterization studies, the lipids1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) and1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC) were alternativelyused as the primary lipid. Lipid films were resuspended in PBS at 2.5mg/ml lipid, and rehydrated by vortexing every 10 minutes for an hour.Lipid suspensions were subsequently extruded through 100 nmpolycarbonate filters using a Mini-Extruder (Avanti Polar Lipids) toobtain monodisperse liposome suspensions. Liposome suspensions werestored at 4° C. and used within a week. To prepare APC-MS formulations,MSRs (10 mg/ml) were incubated with recombinant IL-2 (0.04 mg/ml) in PBSfor 1 hour at room temperature. To form MSR-SLBs, liposomes were addedat lipid:MSR 1:4 (w/w), and incubated for 1 hour at room temperaturewith pipetting every 10 minutes. Next, the material was washed twicewith PBS, and then blocked for 15 minutes by resuspending the materialat 2.5 mg/ml (with respect to MSRs) in 0.25% bovine serum albumin (BSA)in PBS (w/v). Streptavidin, at a molar amount corresponding to 30%theoretical saturation of the amount of biotinylated lipid in theparticular formulation (assuming 34% lipid retention for POPC), wassubsequently added (25 μg streptavidin per 500 μg MSRs for 1%biotinylated-lipid formulations), and the suspension was mixed bypipetting every 5 minutes for 20 minutes. Next, biotinylated T cellactivating cues (1:1 molar ratio TCR-activating cue:αCD28) were added atan amount corresponding to 80% molar saturation of the addedstreptavidin, and the suspension was mixed by pipetting every 10 minutesfor 1 hour. Finally, the material was washed twice with PBS, andresuspended in cell culture media for in vitro assays. APC-MSformulations were used immediately for T cell expansion experiments, orstored at 4° C. and used within a week for characterization studies.

Characterization of MSR-Supported Lipid Bilayer (MSR-SLB) Structure andStability

Brightfield and fluorescence microscopy, used to evaluate MSR lipidcoating, MSR-SLB dispersibility, and MSR-SLB degradation, were performedwith an EVOS FL Cell Imaging System. Confocal microscopy was performedusing a Zeiss LSM 710 confocal system. To evaluate lipid retention withMSRs, MSRs were coated with lipid formulations containing 1 mol %fluorophore-tagged lipid, and lipid retention was quantified using aplate reader. To calculate percent lipid retention over time, culturedmaterial was recovered at specified timepoints by centrifuging at 700rcf for 5 minutes, and fluorescence intensity was normalized to thefluorescence intensity prior to culture. To evaluate MSR-SLB fluidity,fluorescence recovery after photobleaching (FRAP) experiments werecarried out on MSRs coated with lipid formulations containing 1 mol %fluorophore-tagged lipid using a Zeiss LSM 710 confocal system.Photobleaching was performed on the 488 nm laser line and images weretaken every 10 seconds for at least 150 seconds. Fluorescence recoverywas analyzed using ImageJ by normalizing the fluorescence intensitywithin the photobleached region to the fluorescence intensity in anunbleached region on a different rod, at each timepoint.

To quantify IL-2 loading and release, 500 μg of MSRs were loaded with 2μg of IL-2, and then coated with lipid as described. After washing twicewith PBS, IL-2-loaded MSR-SLBs were resuspended in 500 μl release buffer(1% BSA in PBS (w/v)) and incubated at cell culture conditions. Atindicated timepoints, samples were spun down (700 rcf for 5 minutes) andthe supernatants were collected. Subsequently, MSRs were resuspended infresh release buffer and returned to culture. IL-2 in supernatantsamples was quantified via ELISA (Biolegend).

To quantify surface cue loading, MSR-SLB samples were prepared usinglipid formulations containing 0.01, 0.1, or 1 mol % biotinylated lipid.Streptavidin, at an amount corresponding to 30% theoretical saturationof the retained biotinylated lipid (assuming 35% lipid retention forPOPC), was added, followed by the addition of biotinylated IgG at anamount equal to either 40% or 80% saturation of the added streptavidin.As controls, samples containing the same amount of biotinylated IgG butno material were also prepared. All samples were spun at 700 rcf for 5minutes to pellet the material, and the amount of IgG in the supernatantfractions were quantified via ELISA (eBioscience). The biotinylated IgGstock that was used for preparing the samples was also used to preparestandard curves. The amount of IgG loaded onto the material wascalculated by subtracting the amount of IgG detected in control samplesupernatants from the amount of IgG detected in respective materialsample supernatants. For scanning electron microscopy (SEM), cells werecultured with APC-MS on glass coverslips overnight, fixed in 4%paraformaldehyde, and then centrifuged at 2000 rpm for 5 minutes. Fixedsamples were serially transitioned through a gradient of 0, 30, 50, 75,90, 100% ethanol in water. Samples were submerged inhexamethyldisilazane (Electron Microscopy Sciences) and maintained in abenchtop desiccator overnight. Dried coverslips were mounted on SEMstubs using carbon tape, sputter coated with 5 nm of platinum-palladium,and imaged using secondary electron detection on a Carl Zeiss Supra 55VP field emission scanning electron microscope.

In Vitro T Cell Expansion Studies

Polyclonal mouse and human T cell expansion experiments were carried outusing primary CD3+ T cells. Antigen-specific mouse T cell expansionexperiments were carried out using CD8+ T cells isolated from OT-I mice.Antigen-specific human T cell expansion experiments were carried outusing either CD8+ T cells, or PBMCS, isolated from de-identified donorblood samples. Isolated primary mouse or human T cells, or human PBMCs,were mixed with activation stimuli, and cultured for up to two weeks. Inall experiments, non-tissue culture-treated culture vessels were used.For human antigen-specific T cell expansion studies, prior toestablishment of cultures, donor samples were assayed for HLA-A2 MEW Iexpression via FACS, and prior EBV exposure via anti-EBV VCA ELISA (1BLInternational) of serum. Only HLA-A2+ EBV-experienced samples were usedfor expansion studies.

Mock-treated samples in human antigen-specific T cell expansionexperiments were cultured in media supplemented with 30 U/ml recombinantIL-2. Mock-treated samples in all other T cell expansion experimentswere cultured in non-supplemented media. For commercial Dynabeadconditions, DYNABEADS were used according to the manufacturer-optimizedprotocol included with the kit. Briefly, T cells were seeded at adensity of 1×10⁶ T cells/ml with pre-washed DYNABEADS at a bead-to-cellratio of 1:1, in media supplemented with 30 U/ml recombinant IL-2. ForDynabead cultures, 1×10⁵ cells were seeded in the starting culture.Cells were counted every third day and fresh IL-2-supplemented media wasadded to bring the cell suspension to a density of 0.5-1×10⁶ cells/ml.In general, cells were maintained below a density of 2.5×10⁶ cells/mlthroughout the culture period.

For mouse polyclonal studies, APC-MS were prepared that presentedsurface cues (αCD3+αCD28) on between 0.2-1 mol % of the lipids at a 1:1molar ratio, and added into the starting culture at 333 μg/ml. For humanpolyclonal studies, APC-MS were prepared that presented surface cues(αCD3+αCD28) on either 0.1 mol % or 1 mol % of the lipids at a 1:1 molarratio, and added into the starting culture at 33 μg/ml or 333 μg/ml. Formouse antigen-specific studies, APC-MS were prepared that presentedsurface cues (SVYDFFVWL (SEQ ID NO: 3)/H-2K(b) or SIINFEKL (SEQ ID NO:4)/H-2K(b)+αCD28) on either 0.01 mol % or 0.1 mol % of the lipids at a1:1 molar ratio, and added into the starting culture at 33 μg/ml or 333μg/ml. For human antigen-specific studies, APC-MS were prepared thatpresented surface cues (CLGGLLTMV (SEQ ID NO: 1)/HLA-A2 or GLCTLVAML(SEQ ID NO: 2)/HLA-A2+αCD28) on 1 mol % of the lipids at a 1:1 molarratio, and added into the starting culture at 333 μg/ml. APC-MSpresenting cues on 1 mol % of lipids, added at 333 μg/ml, corresponds to˜55 nM of TCR stimuli and αCD28 in the starting culture. For APC msconditions, T cells were seeded with the specified amount of material at5×104 cells/ml in media that was not supplemented with IL-2. In allAPC-MS conditions, 2.5×104 cells were seeded in the starting culture.Media was added throughout the culture period to maintain cells below adensity of 2.5×10⁶ cells/ml. Starting on day 7, when mostmaterial-loaded IL-2 has been released, fresh media that was added wassupplemented with 30 U/ml recombinant IL-2. At specified timepoints,live cells were manually enumerated with a hemocytometer using Trypanblue exclusion, to avoid possible artifacts with automated countingsystems as a result of material contaminants. After enumeration, cellphenotype was evaluated using flow cytometry. Gates were set for eachtimepoint and sample set independently based on fluorescence minus one(FMO) controls.

In Vitro T Cell Functional Studies

For co-culture experiments in which T cell expression of IFNγ and TNFαwas evaluated via intracellular cytokine staining, stimulator cells(mouse, B16-F10; human, T2) were first either unpulsed or pulsed with 1μg/ml peptide (mouse, SIINFEKL (SEQ ID NO: 4); human, CLGGLLTMV (SEQ IDNO: 1) or GLCTLVAML (SEQ ID NO: 2)) for 30 minutes at 37° C.Subsequently, 1×10⁵ expanded cells were cultured with 2×10⁴ stimulatorcells for one hour before adding Brefeldin A (BD Biosciences) to inhibitcytokine secretion, and then cultured for another four hours. Cells werethen stained and analyzed using FACS.

In vitro killing assays were carried out by first incubating targetcells (mouse, B16-F10; human, T2) in 20 μg/ml Calcein AM (Biotium) for30 minutes at 37° C. Target cells were subsequently either unpulsed orpulsed with 1 μg/ml peptide (mouse, SIINFEKL (SEQ ID NO: 4); human,CLGGLLTMV (SEQ ID NO: 1) or GLCTLVAML (SEQ ID NO: 2)) for 30 minutes at37° C. 5×10³ target cells were then cultured with expanded effectorcells at effector cell:target cell (E:T) ratios of 0, 1, 10, 25, or 50for four hours. Cells were then pelleted and the fluorescence intensityof supernatant samples was quantified using a plate reader. IFNγconcentrations in supernatant samples were also quantified via ELISA(Biolegend).

Statistical Analysis

All values were expressed as mean±s.d., unless otherwise specified.Statistical analysis was performed using GraphPad Prism and statisticalmethods are stated in the text. In all cases, p<0.05 was consideredsignificant.

Example 12: Analysis of the Degradation of APC-MS In Vitro

To study the degradation of an exemplary APC-MS in vitro, the followingexperiment was performed. APC-ms (167 μg) comprising αCD3/αCD28antibodies (1% biotinylated lipid) and releasing IL-2 was cultured withprimary mouse T cells (25e⁴ T cells/167 μg MSRs). At various timepoints,cultures were centrifuged at 700 rcf for 5 min, and silica (Si) contentin pellets was quantified via inductively coupled plasma opticalemission spectrometry (ICP-OES; Galbraith Laboratories). As shown inFIG. 37 , silica was undetectable in culture pellets after about 1 week.

Example 13: Controlled Release of Diverse Soluble Immune-DirectingPayloads from APC-MSs

To study the release of a cytokine payloads from exemplary APC-MSs, thefollowing experiment was performed. Four APC-MSs each comprising either2 μg of IL-2, IL-21, TGFβ or IL-15SA were loaded into 500 μg mesoporoussilica micro-rods (MSR) prior to lipid coating. Samples were thoroughlywashed to remove any unloaded protein and subsequently maintained at 37°C. for up to 28 days. Payload release over time was evaluated usingELISA. As shown in FIG. 38 , controlled release of the cytokines fromthe APC-MSs was observed over the course of the experiment. Releasekinetics are likely dependent on physicochemical properties of theparticular cytokine.

Example 14: Conjugation of Antibodies to MSR-SLBs Via Click-ChemistryReaction

To determine whether a functional molecule could be conjugated to theMSR-SLB lipid bilayer the following experiment was performed. IgG wassite-specifically labeled with azide groups using the Thermo SiteClickAntibody Labeling System. MSR-SLBs containing varying amounts (mol %) ofDBCO-modified lipids (Avanti Polar Lipids) were also prepared. As shownin FIGS. 42A and 42B, azide-modified IgG was successfully conjugatedonto the lipid bilayer of MSR-SLBs in a concentration-dependent manner.

EQUIVALENTS

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents to the specificembodiments and methods described herein. Such equivalents are intendedto be encompassed by the scope of the following claims.

INFORMAL SEQUENCE LISTING SEQ ID NO: Amino Acid Sequence 1Cys Leu Gly Gly Leu Leu Thr Met Val 2Gly Leu Cys Thr Leu Val Ala Met Leu 3Ser Val Tyr Asp Phe Phe Val Trp Leu 4 Ser Ile Ile Asn Phe Glu Lys Leu 5His His His His His His 6 Tyr Ile Gly Ser Arg 7Gly Gly Tyr Gly Gly Gly Pro Cys Gly Pro Pro Gly Pro Pro Gly Pro1           5                       10                  15Pro Gly Pro Pro Gly Pro Pro Gly Phe Pro Gly Glu Arg Gly Pro Pro        20                      25                  30Gly Pro Pro Gly Pro Pro Gly Pro Pro Gly Pro Pro Gly Pro Cys    35                      40                  45

1.-95. (canceled)
 96. A method for the manipulation of T cells,comprising contacting an antigen presenting cell-mimetic scaffold(APC-MS) with a biological sample comprising T cells, wherein the APC-MScomprises: high surface area mesoporous silica micro-rods (MSR), whereinspaces between the MSR permit T cell infiltration; a fluid supportedlipid bilayer (SLB) layered on the MSR; and a functional moleculeselected from the group consisting of a T-cell activating molecule, a Tcell co-stimulatory molecule, and a combination thereof, wherein thefunctional molecule is presented on the SLB.
 97. The method of claim 96,wherein the T cells in the sample comprise exhausted T cells.
 98. Themethod of claim 97, wherein the exhausted T cells are CD8+PD-1+ and/orLAG-3+TIM-3+.
 99. The method of claim 96, further comprising detectingthe expression of one or more cell-surface markers in the manipulated Tcells.
 100. The method of claim 99, wherein at least one of the cellsurface markers is selected from the group consisting of CD4, CD8, CD25,CD28, CD36, CD40, CD44, CD45, CD62L, CD69, CD134, FOXP3, 4-1BB, LAG-3,TIM-3, and PD-1.
 101. The method of claim 96, wherein the biologicalsample is obtained from a subject and the APC-MS is contacted with thebiological sample ex vivo.
 102. The method of claim 96, wherein the Tcells are selected from the group consisting of natural killer T cells,gamma delta T cells, CD3+ T cells, CD4+ T cells, CD8+ T cells,regulatory T cells (Tregs), tumor-infiltrating lymphocytes, and acombination thereof.
 103. The method of claim 96, wherein the T cellscomprise Tregs selected from the group consisting of FOXP3+ Tregs,FOXP3-Tregs, and a combination thereof.
 104. The method of claim 96,wherein the T cells are manipulated to generate an expanded populationof effector memory, effector, central memory, and/or naïve T cells. 105.The method of claim 96, wherein the manipulated T cells comprise CD8+cells, CD4+ cells, CD4+/FOXP3− T cells, CD44+/CD62L− T cells, CD8+/CD69+T cells, granzyme B+ CD8+ T cells, and/or IFN-γ-producing T cells. 106.The method of claim 96, wherein the APC-MS comprises a T-cellhomeostatic agent.
 107. The method of claim 106, wherein the T-cellhomeostatic agent is loaded onto the MSR.
 108. The method of claim 96,wherein the APC-MS comprises both the T-cell activating molecule and theT-cell co-stimulatory molecule.
 109. The method of claim 96, wherein theT-cell activating molecule, or the T-cell co-stimulatory molecule, orboth, are presented on the SLB via affinity pairing or chemicalcoupling.
 110. The method of claim 96, wherein the APC-MS comprises animmunoglobulin molecule that binds specifically to an Fc-fusion protein,wherein the immunoglobulin molecule is presented on the SLB.
 111. Themethod of claim 96, wherein the APC-MS further comprises a recruitmentcompound selected from the group consisting of granulocytemacrophage-colony stimulating factor (GM-CSF), chemokine (C-C motif)ligand 21 (CCL-21), chemokine (C-C motif) ligand 19 (CCL 19), Chemokine(C-X-C Motif) ligand 12 (CXCL12), interferon gamma (IFNγ), an FMS liketyrosine kinase 3 (Flt-3) ligand, and a combination thereof.
 112. Themethod of claim 111, wherein the recruitment compound comprises GM-CSF.113. The method of claim 96, wherein the APC-MS comprises an antigenpresented on the SLB.
 114. The method of claim 96, wherein the dryweight ratio of the MSR to the T-cell activating molecule, or the T-cellco-stimulatory molecule, or both if both are present, is between 200:1to 20:1.
 115. The method of claim 96, wherein the spaces between the MSRthat permit T cell infiltration have a mean diameter of 6.8 μm to 12 μm.116. The method of claim 96, wherein the MSR comprise a length of 50 μmto 200 μm.
 117. The method of claim 96, wherein the MSR comprise alength of 80 μm to 120 μm.
 118. The method of claim 96, wherein the MSRcomprise an average length of 100 μm.
 119. The method of claim 96,wherein the MSR comprise an average length of 88 μm.
 120. The method ofclaim 118, wherein the MSR comprise an average diameter of 4.5 μm. 121.The method of claim 96, wherein the MSR comprise an aspect ratio of 20.122. The method of claim 96, wherein the weight ratio of the SLB to theMSR is between 1:4 and 1:20.
 123. The method of claim 96, wherein thebiological sample is a blood sample, a bone marrow sample, a lymphaticsample, or a splenic sample obtained from a human.
 124. The method ofclaim 96, wherein the manipulation results in improved differentiation,expansion, or activity; and/or reduced exhaustion, anergy, or death ofthe T cells.